about crisper cas

1. CRISPR - The Future of Food?
• The Scientific and Agricultural communities have long understood that climate
variability can increase crop failures, potentially causing food shortages.
• Changing and more extreme weather patterns, including drier conditions in
growing regions (which can make crops susceptible to disease and pests), will
continue to negatively impact Global Food security.
• Directed breeding of horticultural crops is essential for increasing yield, nutritional
content, and consumer-valued characteristics such as shape and color of the
produce.
• However, limited genetic diversity restricts the amount of crop improvement that
can be achieved through conventional breeding approaches.
• It is believed that CRISPR can have a positive impact on Food Productivity,
Quality, and Environmental sustainability.

2. • Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement
through the introduction of genetic variation in a targeted manner.
• The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-
engineering) provides a more refined method for modulating gene expression and
creating phenotypic diversity to benefit crop improvement.
• CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural
crops that are better able to adapt to climate change and providing food for an
increasing world population.
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3. UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT
COLLEGE OF HORTICULTURE, BENGALURU
VSC 504 (2+1)
Presentation on
CRISPR – CAS & ITS UTILIZATION IN VEGETABLE CROPS
PRESENTED TO:
Dr. C. N. Hanchinamani
Professor and Head
Dept. of Vegetable Science
PRESENTED BY:
Saloora Abhijeeth
Jr. M.Sc.
Dept. of Vegetable Science
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4. CRISPR CAS???
• CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats
CAS - CRISPR-Associated nucleases Systems or CRISPR Associated Proteins
• Prokaryotic Adaptive Immune System (Antivirus Mechanism)
• Represented in most Archaea and many Bacteria.
• Among the currently known prokaryotic defense systems, the CRISPR-CAS genomic
loci show unprecedented complexity and diversity.
• Three major types of CRISPR-Cas systems are at the top of the classification
hierarchy.
• The 3 types are readily distinguishable by the presence of three unique signature
genes: Cas3 in type I systems, Cas9 in type II, and Cas10 in type III
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5. Where did it Start?
• IN 1987, CRISPR/CAS system was discovered after identification of similar DNA
sequences in the genome of Escherichia coli while studying genes that are helpful in
phosphate metabolism.
• Later on, these sequences have been identified in other bacterial genomes including
Halophilic Archaea.
• These sequences play an important role in evolutionary relationship of an organism.
• In Hyper Thermophilic Archaea, it is hypothesized that CAS protein is involved in
the DNA repairing mechanism.
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6. How does this mechanism work in Prokaryotes?
• In most Archaea and many Bacteria, they function on the self - non self
discrimination principle.
• These systems incorporate fragments of Alien DNA (known as Spacers) into
CRISPR cassettes.
• Then transcribe the CRISPR arrays including the spacers, and process them to
make a guide crRNA (Guide CRISPR RNA or gRNA).
• Employed to specifically target and cleave the genome of the Cognate Virus or
Plasmid.
• Numerous, highly diverse Cas (CRISPR-associated) proteins are involved in
different steps of the processing of CRISPR loci transcripts, cleavage of the
target DNA or RNA, and new spacer integration.
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8. • The action of the CRISPR-Cas system is usually divided into three
stages:
(1) Adaptation or Spacer integration
(2) Processing of the primary transcript of the CRISPR locus
(precrRNA) and maturation of the crRNA which includes the spacer
and variable regions corresponding to 5′ and 3′ fragments of CRISPR
repeats, and
(3) DNA (or RNA) interference.
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9. 1. ADAPTATION PROCESS
• Two proteins, Cas1 and Cas2 – that are present in the great majority of the known
CRISPR-Cas systems - Sufficient for the insertion of spacers into the CRISPR cassettes.
• These two proteins form a complex that is required for this Adaptation Process.
• The endonuclease activity of Cas1 is required for Spacer integration whereas Cas2
appears to perform a Non enzymatic function.
• The Cas1-Cas2 complex represents the highly conserved “information processing”
module of CRISPR-Cas that appears to be quasi autonomous from the rest of the
system.
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10. 2. PROCESSING
• The processing of pre-crRNA into the guide crRNAs, is performed either by a
dedicated RNA endonuclease complex or via an alternative mechanism that
involves bacterial RNase III and an additional RNA species.
• The mature crRNA is bound by one (type II) or several (types I and III) Cas
proteins that form the effector complex, which targets the cognate DNA or RNA.
• The effector complex of type I systems is known as Cascade (CRISPR-associated
complex for antiviral defense).
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11. 3. INTERFERENCE OF DNA / RNA:
• The CAS & crRNA complex recognise the cognate virus DNA and break those
strands apart.
• Hence, the viral genome is inactivated.
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14. CRISPR – CAS9 SYSTEM:
• Due to its simplicity and efficiency, it has rapidly become the most widely used
tool for editing animal and plant genomes.
• Ideal for modifying the traits of many plants, including food crops, and for
creating new germplasm materials.
• Has been in various monocot and dicot plants to
i. enhance yield and nutrition value
ii. to introduce or enhance tolerance to biotic and abiotic stresses.
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15. • Nobel Prize in Chemistry 2020 was awarded jointly to two women scientists and an
all-women team, France’s Emmanuelle Charpentier and the US’s Jennifer Doudna,
for discovering one of gene technology's sharpest tools: the CRISPR/Cas9 genetic
scissors.
• CRISPR/Cas9 is considered to be a revolutionary finding and one of the most exciting
innovations primarily for being precise and the sharpest tool for gene editing.
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16. MECHANISM
• A typical class 2 type II CRISPR system, is widely applied at present.
• In 2013, the type II CRISPR system was first isolated from Streptococcus pyogenes
(SpCas9).
• The trans-activating crRNA (tracrRNA) base pairs with the repeat sequence in the
crRNA to form a unique dual RNA hybrid structure guide
• This directs Cas9 to cleave the target DNA, so a chimeric sgRNA was designed that
combines crRNA and tracrRNA into a single RNA transcript, simplifying the system
while preserving Cas9-mediated full-function sequence-specific DNA cleavage.
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17. • Contains two nuclease domains, RuvC and HNH,
which cut the target DNA strands and non-target
DNA strands respectively.
• A short trinucleotide protospacer adjacent motif
(PAM) - Essential for initial target sequence
recognition.
• The target sequence could not be recognized
without a corresponding PAM site.
• After successful identification, a double-strand
break (DSB) occurs upstream of the 3′-NGG PAM
site.
• This system - being widely used in
i. Genome editing
ii. Single-nucleotide-mutation detection
iii. and other fields
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SOURCE: DAS et al., 2023

19. Genetically Modified (GMOs) Gene Edited (CRISPR)
Less accurate - the change is initiated in a random
location in the genome
Highly accurate - targeted gene editing in the
exact spot where a change is needed in the genome
DNA can be exotic - the genes placed in the
genome may be synthetic or taken from another
species
DNA is native - DNA that is already a part of the
organism is removed (cut out) or altered (edited)
You transfer genes from one species to another to
endow the organism with pest resistance
You rewrite an organism’s genetic code to make it
less susceptible to pests
The alteration would never have happened
naturally through evolution Example: A cow
developing wings and learning to fly
The alteration could have occurred naturally
through evolution Example: A cow developing
resistance to tuberculosis
Highly expensive - only larger companies can
benefit
Extremely cost effective - small farming
operations can utilize the technology
GMO vs CRISPR
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Applications in
Vegetable Crops

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Strategies for applying CRISPR/Cas-mediated cis-engineering in horticultural crops

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Vegetables
modified with
CRISPR
APPLICATIONS
SOURCE: DAS et al., 2023

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Share of different Horticultural and Agricultural Crops in which CRISPR mechanism is used
Source: Lie et al. Horticulture Research (2020)

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APPLICATIONS The GABA-enriched Tomato: The world's first CRISPR-edited crop
• The CRISPR-Cas9 system was first used in tomatoes in
2014.
• Scientists used the technology to knock out Argonaute 7,
which led to wiry phenotypes. A Sicilian rouge tomato
was used for the purpose.
• In 2021, a tomato became the very first genome-edited
food made with CRISPR–Cas9 technology to be available
on the market.
• The motivation behind increasing GABA (γ-aminobutyric
acid) in this fruit is that research has shown its impact on
lowering blood pressure and promoting relaxation.
• Launching the world's first CRISPR engineered food
product, it is an important milestone.
Source: www.azolifesciences.com

25. 1. Deletions and inversions
Cucumber
• A 39.9-kb deletion and a 16.2-kb deletion located 16.5-kb upstream of cucumber
FLOWERING LOCUS T (CsFT) are both associated with higher CsFT expression
levels and earlier flowering.
• The CsFT locus was selected during cucumber domestication and has been
important in its adaptation to higher latitudes for cultivation.
Tomato
• One of the remarkable examples of variation in locule number is controlled by a
nearly 300-kb inversion of the fasciated (fas) locus in tomato.
• The fas locus is characterized by disruption of the promoter region of tomato
CLAVATA3 (SlCLV3), leading to downregulation of the gene and larger fruit with
increased number of locules.
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Applications using CRISPR/Cas mechanism
Source: Lie et al. Horticulture Research (2020)

26. 2. Insertions
Insertions are sources of genetic diversity that can alter gene expression by
introducing new or disrupting existing CREs. Especially transposable elements (TEs)
play important roles in creating genomic variation by altering gene regulation.
• In Cauliflower (Brassica oleracea var botrytis), a 695-bp Harbinger DNA
transposon insertion in the MYB2 promoter increases the expression of this gene,
resulting in a purple phenotype.
• Additionally, the differentiation of winter and spring genotypes in rapeseed
(Brassica napus L.) primarily arose from a MITE transposon insertion in the
upstream region of BnFLC.A10.
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Source: Lie et al. Horticulture Research (2020)

27. 3. Epigenetic variations
• Natural epigenetic variations contribute to heritable phenotypic diversity that is
not caused by modification in the DNA sequence.
Tomato
• One of the best examples of an epiallelic variant that impacts an important
agronomical trait is the Colorless Non-Ripening (Cnr) allele in tomato.
• The epiallele of LeSPL-CNR is responsible for colorless fruits with a substantial
loss of cell-to-cell adhesion.
• In Cnr mutants, hyper-methylation was found along a 286-bp CRE located ~2.4-
kb upstream from the first ATG of LeSPL-CNR.
• This change in methylation status likely explains the reduced expression level of
LeSPL-CNR and the ripening defects in Cnr fruits.
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Source: Lie et al. Horticulture Research (2020)

28. 4. Promoter insertion/swapping
• Promoter insertion and swapping can be achieved by homology-directed
repair (HDR) with potentially great importance to crop improvement.
• However, HDR has been challenging due to its low efficiency in higher
plants.
• A 35S promoter was inserted upstream of anthocyanin 1 (ANT1), resulting in
enhanced anthocyanin accumulation and intensely purple tomato tissues.
• Additionally, glyphosate tolerant cassava (Manihot esculenta) was generated
by a promoter swap of the 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS) gene.
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Source: Lie et al. Horticulture Research (2020)

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Source: Lie et al. Horticulture Research (2020)

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Source: Lie et al. Horticulture Research (2020)

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Application of the CRISPR/Cas9 system to enhance vegetable crop yield, nutritional quality, and
other agronomic traits
Crop Target
Gene (s)
Mutation Mode of delivery Trait
modified
Key
observations
References
Cabbage
(Brassica
oleracea var.
capitata L.)
PDS Knockout Agrobacterium
tumefaciens strain
EHA105 mediated
hypocotyl
transformation
Phenotype - Ma et al. (2019)
Cucumber
(Cucumis
sativus L.)
elF4E Knockout Agrobacterium‐mediated
cotyledon transformation
Virus
resistance
Resistance
against
Cucumber vein
yellowing virus,
Zucchini yellow
mosaic virus,
PRSMV - W
Chandrasekaran
et al. (2016)
Eggplant
(Solanum
melongena L.)
SmelPPO1‐
10
Knockout A. tumefaciens strain
LBA4404
mediated transformation
Enzymatic
browning
Browning ↓ Maioli et al.
(2020)
SOURCE: DAS et al., 2023

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Kale (Brassica
oleracea var.
alboglabra)
BoaCRTISO Knocked
down
A. tumefaciens strain
GV3101 mediated
transformation
Color Carotenoid and
chlorophyll
biosynthesis ↓
B. Sun et al.
(2020)
Tomato
(Solanum
Lycopersicum
L.)
ANT1 Knockout A. tumefaciens mediated
transformation
Color Anthocyanin ↑ Čermák et al.
(2015)
RIN Knockout A. tumefaciens mediated
method
Fruit
ripening
Red color
pigmentation in
mutant ↓
Ito et al. (2015)
DMR6 Knockout A. tumefaciens strain
GV3101 mediated
cotyledon transformation
Disease
resistance
Resistance against
Pseudomonas
syringae pv. tomato
and Phytophthora
capsica,
Xanthomonas spp
Paula de
Toledo
Thomazella
et al. (2016)
SOURCE: DAS et al., 2023

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Tomato
(Solanum
Lycopersicum L.)
Mlo 1 Knockout A. tumefaciens
mediated cotyledon
transformation
resistance
Disease
resistance
Resistance
against Oidium
neolycopersici
Nekrasov et
al. (2017)
GAD Knockout A. tumefaciens
mediated
transformation
Quality
improvement
GABA ↑ Nonaka et al.
(2017)
G3P,
DXS,
GGPPS,
PDS,
ZISO
Knockout A. tumefaciens
mediated
transformation method
Phenotype Lycopene
content ↑
Li, Wang, et
al. (2018)
lncRNA14
59
Knockout A. tumefaciens
mediated
transformation
Fruit ripening Ethylene ↓,
lycopene↓
R. Li, Fu, et
al. (2018)
SOURCE: DAS et al., 2023

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Potato
(Solanum
tubersoum L.)
StALS1 Knockout A. tumefaciens
mediated
transformation
Herbicide
resistance
- Butler et al.
(2015)
St16DOX Knockout A. rhizogenes strain
ATCC15834 mediated
transformation
- 22,26‐dihydroxy
cholesterol ↑
Nakayasu et
al. (2018)
StPPO2 Knockout - Enzymatic
browning
PPO ↓ González et
al. (2020)
Pumpkin
(Cucurbita
moschata
Duchesne)
RBOHD Knockout A. rhizogenes mediated
transformation
Salt tolerance H2O2 ↓, K+↓ Huang et al.
(2019)
Sweet potato
(Ipomoea
batatas (L.)
Lam.)
GBSSI,
SBEII
Knockout A. tumefaciens strain
LB4404
mediated
transformation
Quality
improvement
Amylose ↓,
amylopectin ↓
H. Wang et
al. (2019)
SOURCE: DAS et al., 2023

35. • Relatively New Genome-Editing Technology.
• Allows researchers to more easily alter DNA sequences and modify gene
function in animals or plants (within the genome alteration).
• Allows scientists to make targeted changes in the genome of an organism, be it
insertion, deletion, modification or replacement of gene sequences.
• The key strength of CRISPR-based breeding - It allows for faster and more
targeted development of crop varieties.
• Traditional methods take between seven and ten years (if possible at all), they can
now be done within two to four years.
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ADVANTAGES

36. LIMITATIONS
• Toxicity and Immunogenecity of CAS Proteins.
• If a genome sequence is unavailable or unassembled, it is impossible to identify
potential targets of interest for editing or assess off – target activity of gRNAs.
• Undesired Mutations due to non – target effects.
• Variable effeciencies of gRNA
• Large protein size leads to difficulty in gene delivery which in turn hinders editing
efficiency.
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CASE STUDY

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Targeted creation of new mutants with compact plant architecture using
CRISPR/Cas9 genome editing by an optimized genetic transformation
procedure in cucurbit plants
Tongxu Xin et al., 2022
OBJECTIVE: To establish an efficient genetic transformation system for melon and
squash using an “optimal infiltration intensity”.
LOCATION: Key Laboratory of Biology and Genetic Improvement of Horticultural
Crops of Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Beijing,
China.
Source: Horticulture Research, 2022 ( NAAS Score: 13.29)

39. Materials and Methods
1. Media used in the study:
• GM (germination medium): 6-benzylaminopurine (BA, 2 mg/L for Cu2, 404 and Eu1, 0.5 mg/L for melon
and Xintaimici, 1 mg/L for jingxinzhen No. 4 and jingxinzhen No. 11) and 1 mg/L abscisic acid (ABA) were
added to MS medium.
• IM (inoculation medium): BA (2 mg/L for Cu2, 404 and Eu1, 0.5 mg/L for melon and Xintaimici, 1 mg/L
for jingxinzhen No. 4 and jingxinzhen No. 11), 1 mg/L ABA, 200 μM acetosyringone (AS) and 1.25 M
morpholioethanesulfonic acid (MES) (pH 5.2) were added to MS medium.
• COM (co-cultivation medium): BA (as in IM, above), 1 mg/L ABA, 200 μM AS, 1.25 mM MES (pH 5.2),
and 250 μM LA were added to MS medium.
• SIM (shoot induction medium): BA (as in IM, above), 2 mg/L AgNO3, 1 mg/L ABA and 200 mg/L
Timentin were added to MS medium.
• RIM (root induction medium): 200 mg/L Timentin and 2 mg/L AgNO3 were added to MS medium. LB
(liquid medium for Agrobacterium culture): LB liquid medium with kanamycin (50 mg/L) and rifampicin
(25 mg/L).
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40. 2. Plant materials and growth conditions
• Ten melon varieties were analyzed in this study: P147 (cultivated melo), ivf105
(cultivated agrestis), m1 (cultivated melo), m2 (cultivated melo), m3 (cultivated
melo), m4 (cultivated melo), m5 (wild melo), m6 (cultivated melo), m7 (cultivated
agrestis) and Jingyu (commercial hybrid), of which ivf105, and m1 to m7 were
provided by Huaisong Wang of the Institute of Vegetables and Flowers (IVF), Chinese
Academy of Agricultural Sciences.
• The seeds of cucumber inbred line Cu2 (South China type), Xintaimici (Northern
China type), 404 (Northern China type) and Eu1 (European-type cucumber) were
used in this study.
• Two commercial moschata materials, jinxinzhen No. 4 and jigxinzhen No. 11, were
used in squash transformation.
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41. • All tissue cultures were maintained in a culture room at the differentiation and rooting
stages.
• After rooting, the regenerated plants were transferred into an incubator for about a
week and planted in a greenhouse under a light regime of 16 h light/8 h dark at a
temperature ranging from 180C to 240C.
• Seeds were soaked in warm, distilled water at 500C for 30 min, and the seed coat was
removed.
• Surface sterilized with 75% ethanol for 15 s and 1% sodium hypochlorite solution for
15 min, then rinsed six times in sterile distilled water.
• Sterilized seeds of melon, squash, and cucumber were spread on Petri dishes
containing 7.5 mL sterilized water for 1–2 days at 28◦C.
• Only the Cu2 seeds were spread on Petri dishes containing GM medium at 28◦C.
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42. 3. sgRNA design and plasmid construction:
• The sgRNA was designed using the CRISPR-GE web tool (http://skl.scau.edu.cn).
• The sgRNA was inserted into the pBSE402 vector.
• In brief, a 50-μL mix containing 1.5 μL 100 μM forward primers, 1.5 μL 100 μM
reverse primers, 5 μL 10× NEB buffer 3.1, and 42 μL water was incubated at 95◦C
for 5 min, and the temperature was then decreased by 0.1◦C/s to 20◦C to produce a
short double-stranded DNA fragment.
• The short DNA fragment was inserted into pBSE402 using the restriction enzyme
BsaI and T4 Ligase.
• The constructed recombinant vector was transformed into Agrobacterium strain
EHA105.
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43. 4. Agrobacterium-mediated transformation
• Two days before transformation, Agrobacterium stock cells (EHA105) carrying
the vector were shaken (200 rpm) using 3 mL liquid LB medium with 50 mg/L
kanamycin and 25 mg/L rifampicin for 24 h at 28◦C.
• The starter cultures were transferred to 50 mL liquid LB medium in a 1:1000 ratio
and cultured overnight to OD600 0.6–0.8.
• The Agrobacterium culture was centrifuged and resuspended to OD600 0.2 with
IM medium.
• Explants were always kept on wet filter paper.
• The treated explants were transferred to a 100-mL triangular bottle containing 20
mL Agrobacterium suspension.
• The bottle containing the explants and Agrobacterium suspension was sonicated at
100 W using an Ultrasonic cleaning instrument (KQ-100DV) different numbers of
times (depending on the optimal infection conditions).
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44. • The explants were cultured on SIM for 3–4 weeks.
• Explants with GFP fluorescent shoots were selected and transferred to jars
containing RIM for rooting.
• Rooted seedlings were planted in the soil to promote growth and development.
5. Detection of GFP fluorescence:
• To screen the GFP-positive plants, explants regenerated for one month were
examined using a Leica MZ10 F stereomicroscope (Leica Microsystems,
Germany) at the tissue culture stage.
6. GFP intensity calculation
• The GFP expression level of the explants after co - cultivation was calculated
using Image J.
7. Detection of targeted mutations
8. Detection of editing efficiency with Hi-TOM sequencing
9. Off target analysis based on whole-genome resequencing (WGS): The
sequenced data were analysed using the CRISPResso algorithm.
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45. 45
Overview of the eight-stage procedure for Agrobacterium-mediated transformation
(a) Removal of seed coat, sterilization, and
germination.
(b) Preparation of Agrobacterium tumefaciens,
culturing in LB liquid medium, transfer, and
shaking to OD = 0.6–0.8.
(c) Explant preparation, excision of
embryos from the germinated seeds, cutting of
cotyledons in half transversely, and selection of
proximal parts with U-shaped ends as explants.
(d) Infection, scratching of the proximal regions of
explants with a micro-brush, and sonication.
(e) Vacuum infiltration. Vacuum treatment was
applied with a syringe.
(f) Co-cultivation with A. tumefaciens in the dark.
(g) Shoot regeneration and screening. The dark-
cultured explants were washed
with sterilized water, then transferred to shoot
induction medium. Explants regenerated for about
4 weeks and were then screened for positive buds
by fluorescence microscopy.
(h) Root regeneration. The positive buds were
removed and transferred to rooting medium.

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(a–d) Schematic view of sgRNA target sites and
editing results for the ER gene in melon,
cucumber, and squash. The target sequences are
highlighted in red and underlined, and the PAM
sites are highlighted in bold and underlined.
(e) T-DNA region of the pBSE402 vector.
(f–i) Genome editing results of T0 generation
plants. sgRNA and protospacer-adjacent motif
(PAM) sequences are highlighted by red and bold,
respectively. Black dash indicates deletion. Blue
letters indicate insertion. The text on the right
indicates the editing type. The numbers in
parentheses represent the proportion of this
editing type in the total reads.
(j) The columnar stacking diagram shows the
target mutation rate based on Hi-TOM analysis
(reads of target mutation/total reads of target site).
Different colors represent different mutation
types. D indicates deletion, and I indicates
insertion
Vector, sgRNA map, CRISPR/Cas9 editing target sites, and editing efficiency.

47. Results & Discussions
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The optimal infiltration intensity strategy for genetic transformation of melon
• To establish a genetic transformation system for melon, we first tested ten melon
varieties to identify those with higher regeneration efficiency.
• For each variety, 80–110 explants were cultured in regeneration medium for about
one month with three biological replicates.
• The regeneration rate was calculated as the percentage of explants with
regenerated shoots divided by the total number of explants.

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(a) Shoot regeneration ratio of ten different melon varieties. The red star
indicates that the highest rate was 93.8% for m1. Different lowercase
letters indicate significant differences (p < 0.05, Tukey’s test). (b) The
cotyledon explant of melon. The curved white line represents the U-
shaped cut end. The white dash represents the cross section of panel c. (c)
Cross-section along the white dash of panel b, showing that the cambium
cells are located in the deeper layer of vascular tissue. (d) From left to
right, each column represents different infection treatments and effects.
V: vacuum, B: micro-brush, S: sonication. The first line shows the
cotyledon explants of melon after co-cultivation with Agrobacterium
tumefaciens in the dark. From the second line to the sixth line,
examination of GFP fluorescence after co-cultivation showed the region
and intensity of the fluorescent signal. The third and fourth lines show the
infected areas in longitudinal section. The fifth and sixth lines show the
infected areas in cross-section. (e, f, and g) GFP intensity, explants with
infected vascular tissue, and survival rate under four different treatments,
respectively. Data are means of three replicates, and error bars indicate
standard deviations. Different lowercase letters indicate significant
differences (p < 0.05, Tukey’s test).
Increasing infection intensity is the key to genetic
transformation of melon

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(a, b and c) GFP intensity, explants with infected
vascular tissue, and survival rate of melon m1
under five different treatments, respectively.
Data are means of three replicates,
and error bars indicate standard deviations.
Different lowercase letters indicate significant
differences (p < 0.05, Tukey’s test). (d) A
regenerated transgenic T0 plant of melon was
transferred to the soil. The GFP-fluorescent
apical meristem (e) and seeds (f and g) of the
transgenic melon T0 plant. The transgenic T1
plant (h) and WT (i) of melon under the GFP
channel of the LUYOR-3415RG. Tissues appear
green because of GFP expression and red
because of chlorophyll autofluorescence.
The genetic transformation system of melon was established
using the strategy of optimal infiltration intensity

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The genetic transformation system of squash was established
using the strategy of optimal infiltration intensity
(a, b and c) GFP intensity,
explants with infected vascular tissue, and
survival rate of squash jingxinzhen No. 4
under five different treatments,
respectively. Data are means of three
replicates, and error bars indicate standard
deviations. Different lowercase letters
indicate significant differences (p < 0.05,
Tukey’s test). (d) A
regenerated transgenic T0 plant of squash
after one week of transfer to soil. The GFP-
fluorescent tendril (e), apical meristem (f),
and seeds (g) of a
transgenic squash T0 plant. A transgenic T1
plant (h) and WT plant (i) of squash under
the GFP channel of the LUYOR-3415RG

52. The optimal infiltration intensity strategy an antioxidant application for genetic
transformation of squash
• They selected two commercial squash varieties, “jingxinzhen No. 4” and “jingxinzhen
No. 11” with high regeneration efficiency (87.6% and 78.4%, respectively).
• Explants of “jingxinzhen No. 4” and “jingxinzhen No.11” were exposed to sonication
ranging in duration from 0 to 20 s in combination with micro-brushing and vacuum
infiltration of Agrobacterium.
• We observed a high mortality rate among explants after sonication, even at a duration
of only 5 s.
• This result underscores the low tolerance of this cultivar to wounding.
• Agrobacterium-mediated infection is well known to impose stress on plant cells, thus
affecting plant tissue survival and regeneration.
• Several antioxidants, such as silver nitrate (AgNO3), cysteine (Cys), dithiothreitol
(DTT), polyvinylpyrrolidone (PVP), and lipoic acid (LA) can reduce browning and
death of plant tissues when added to the culture medium.
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Conclusion:
• Three of the cultivars of Melon had a regeneration efficiency greater than 80%.
Among these, the cultivar “m1” had the highest regeneration efficiency (93.8%)
and was therefore used for further investigations.
• Under Squashes, “jingxinzhen No. 4” and “jingxinzhen No. 11” with high
regeneration efficiency (87.6% and 78.4%, respectively).
• These results indicate that the optimal infiltration intensity strategy effectively
improves the genetic transformation efficiency of cucumber and partially
overcomes the classic issue of genotype dependence.
• This methodology may be compatible with other cucurbit crops, as well as other
species

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