CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

Vasantrao Naik Marathwada Krishi Vidyapeeth,
Parbhani
College of Agriculture, Parbhani
Akshay Deshmukh Ph.D. Scholar (2019 A/05P)
1
CRISPR/Cas9 Genome Editing Tool in Plant Breeding
Name of Student : Deshmukh A. S. Registration No. : 2019A/05P
Research Guide : Dr. D. B. Deosarkar Seminar In-charge : Dr. H.V. Kalpande
Semester : III Date : 01/04 /2021
Course No : GP-691 Course Title : Doctoral Seminar
Research Guided:-
Dr. D. B. Deosarkar
Associate Dean and Principal
College of Agriculture,
Golegaon
Head of Department:-
Dr. J. E. Jahagirdar
Dept. Agril. Botany
Parbhani
Seminar Incharge:-
Dr. H. V. Kalpande
Department of Agril. Botany
Parbhani
Presented by – A. S. Deshmukh Ph. D. Scholar (201A/05P)

2
1 • Genome Editing
2 • CRISPR/Cas9
3 • History of CRSPR/Cas System
4 • Prerequisites of CRISPR/Cas System
5 • Target genome editing using CRISPR/Cas9
6 • Mechanism of CRISPR/Cas9 System
7 • Types of CRISPR/Cas9 system
8 • Applications of CRISPR/Cas9 System
9 • Limitation of CRISPR/Cas9 System
10 • Improvement to CRISPR/Cas9 System
11 • Future recommendation of CRISPR/Cse9 System
12 • Case Study
13 • Conclusion of Seminar
INDEX

Genome Editing
 Genome editing is a group of technologies that gives
scientist the ability to change an organism’s DNA.
 These technologies allow genetic material to be added
removed or altered at particular location in the genome.
 Why Genome editing…..?
 To understand the function of gene or a protein, one
interferes with its in sequence-specific way and monitors
its effects on the plants phenotype.
 Allow site specific mutagenesis.
 Genome editing gives cent percent results.
Genome editing work on Site specific Nuclease mechanism 3

Site-Specific Nucleases(SSNs)
4
Nonspecific
Endonucleases
(have the ability to produce
DSBs in DNA)
DNA-binding domains
proteins
(specifically bind to DNA sequences
that are complementary to Specific
Sites)
Jaganathan et al. (2018)

“knocking in” “knocking out”
.
After the DNA break is induced, it will trigger the DNA repair mechanisms.
 Non-homologous end joining (NHEJ) mechanism.
 Homologous-directed repair (HDR) mechanism.
 SSNs-based genome editing system is classified into three categories
Leong et al. (2018) 5

SSNs
Ist Generation
TALEN
Zinc Finger
2nd
Generation
CRISPR/Cas9
Why CRISPR/Cas9……..?
 Target Design Simplicity
 Efficiency
 Multiplexed mutation
Leong et al. (2018) 6

CRISPR/Cas9
 Clustered regularly interspaced short palindromic repeats
(CRISPR)/ CRISPR-associated nuclease 9 (Cas9)
 CRISPR is a family of DNA sequences found in
the genomes of prokaryotic organisms such as bacteria.
 These sequences are derived from DNA fragments
of bacteriophages that had previously infected the
prokaryote.
 They are used to detect and destroy DNA from similar
bacteriophages during subsequent infections.
 The CRISPR-Cas system is a prokaryotic immune system
that confers resistance to foreign genetic elements such as
those present within plasmids and phages and provides a
form of acquired immunity
 Cas9 is a 160 kilodalton protein which plays a vital role in
the immunological defense of certain bacteria
against plasmids.
7
Wikipedia

History of CRISPR/Cas System
1987
• CRISPR repeats first observed in bacterial genome.
2002
• CRISPR elements and associated genes identified and named.
2005
• CRISPR Spacer identified foreign DNA.
2006
• CRISPR proposed to be a bacterial adoptive immune system.
2012
• CRISPR/Cas9 developed as gene editing tool.
2013
• First use of CRISPR/Cas9 in plants.
2015
• CRISPR/Cas9 used to develop virus-resistant tomato plants.
2016
• USDA determines CRISPR/Cas9 edited crops will be not be regulated as GMOs.
2017
• US patent office awards key CRISPR/Cas9 patents to the broad institute.
2020 • Jennifer A. Doudna & E. Charpentier awarded a Nobel Prize for valuable work on CRISPR.
8
www.sciencedirect.com

9
Noble Prize Winner Jennifer Doudna Emmanuelle Charpentier

Prerequisites of CRISPR/Cas
System
10

CAS-9 Nuclease
11
HNH domain
• cleaves the target DNA
strand complimentary to the
guided RNA sequence
RuvC domain • cleave the non target strand
El-Moundai et al. (2020)

Mechanism of CRISPR/Cas9 System
 Formation of the editing Complex
 Cas9 pairing with sgRNA
 sgRNA carry complimentary sequences and deliver Cas-9 to the genome
12

 Pairing with the target gene
 Find complimentary sequences
 Cas9 + sgRNA join the target genome
13

Cutting the Target DNA
 Cas9 cut the target gene on the genome
 The all attempts to repair the DNA that creates a mutation
that disables its function permanently
14

Inserting new gene
 Desirable gene with a specific
function is then inserted to fill the
gap and replace the original gene
15

 Production of the desired protein
 New gene is ready
16

.
17
Pipeline of
CRISPR/Cas9
Genetic Transformation
of Genes
Manghwar et al. (2019)

Methodology's for the screening of
CRISPR/ Cas9 Plants
 qPCR
 Mutated DNA sequence easily determine by PCR
 qPCR can be used to distinguish wish homozygous and Heterozygous mutation.
 Surveyor Nuclease and T7 endonuclease assays
 Widely use
 Recognize and digest mismatched heteroduplex DNA.
 It identify the mismatch and cleave it downstream to the mismatch
 High-resolution melting analysis (HRMA) based assays
 DNA sequence amplification by qPCR
 Incorporating fluorescent dye followed by amplicon melt curve analysis
 High-Throughput Tracking of Mutation (H-TOM)
 Hi-TOM is an software for detection of mutation cause by the CRISPR system.
 Whole-genome sequencing (WGS) to detect on- and off- target
 It is crucial to understand the scope of on and off target mutation
18

Types of CRISPR/Cas9 System
20
Jennifer Doudna (2018)
Class-1 Class-2
Type-I Type-III Type-II Type-V Type-VI
cascade Csm/emr Cas9 Cas12 Cas13
Non intrinsic nuclease
activity in cascade
recruits Cas3 to
cleaves DNA
Csm cleaves DNA
(transcription dependent)
RNA
Csm6 is an auxiliary
RNase
Cleaves
dsDNA
Cleaves
dsDNA
Cleaves
ssDNA

Different Cas proteins and their
functions
Protein Distribution Process Function
Cas1 Universal Spacer acquisition DNAse, Not sequence specific, can bind RNA; Present
in all types
Cas2 Universal Spacer acquisition Specific to U rich region; Present in all types
Cas3 Type I Signature Target Interference DNA helicase, Endonuclease
Cas4 Type I, II Spacer acquisition RecB- Like nuclease with endonuclease activity
homologous to RecB
Cas5 Type I Spacer acquisition RAMP protein, endoribonuclease involve in crRNA
biogenesis; Part of CASCADE
Cas6 Type I, III Spacer acquisition RAMP protein, endoribonuclease involve in crRNA
Cas7 Type I Spacer acquisition RAMP protein, endoribonuclease involve in crRNA
Cas8 Type I Spacer acquisition Large protein with McrA/HNH-nuclease domain &
RuvC- like nuclease; part of CASCADE
Cas9 Type II Signature Target Interference Large multidomain protein with McrA-HNH
nuclease domain & RuvC- like nuclease domain;
necessary for interference & target cleavage
Cas10 Type III Signature crRNA Expression &
Interference
HD nuclease domain, palm domain Zn Ribbon, some
homologies with CASCADE elements. 21

Applications of CRISPR/Cas9
System
 Gene Silencing
 By Gene Knockout
 Transcriptome Analysis/Modification
 Allow Selective Transcription
 By shutdown the Cas9 protein in the CRISPR/Cas9 and adding
transcription factor.
 Targeted mutagenesis
22
Crop Knockout gene Gene Function Achievement
Maize Waxy (WX1) Starch-Synthesis Protein
that involved in Kernel
Maintenance
Uniformity
Stability

 Genome-edited Plant
 Paint Genome
By deactivating PAM site or catalytic subunit of Cas9, and adding tag FP
(Fluorescent protein) to mark location of specific gene in our genome.
 Epigenetic modification
 Gene replacement and gene Knock-in
 Eg. Replacement of the endogenous 5-enolpyruvylshikimate-3-phosphate synthase
(OsEPSPS) in rice with a gene encoding a form of the protein tolerant to the
herbicide glyphosate.
 Multiple GE
Crop Trait improved Name of Organization
Waxy Corn Disease and Drought resistance DuPont Pioneer (USA)
Wheat Produce gluten-free wheat by
eliminating gliadins in wheat
Institute for Sustainable
Agriculture (Spain)
Soybean Produce healthier oil with reduced
unsaturated fat content by increasing
the percentage of oleic acid
Institute for Basic Research
(IBS) (South Korea)
White button
mushroom
Browning resistant Yinong Yang; Penn State
College of Agricultural Science
23

Limitation of CRISPR/Cas9
System
24
Bigger protein size
Limited PAM size
Low HDR efficiency
It introduce multiple and random
mutations in the genome
Needs Agrobacterium-mediated
transformation system
Misuse
Hard to commercialize the
transgenic crops

25
Dr. He Jiankui
World’s First Genetically Modified Babies
He had worked toward this for two years, altering their
genes as embryos to try making them resistant to their
father’s HIV infection.

Improvements to CRISPR/Cas9 System
 several modifications of the Cas9 enzyme have
been developed to increase target specificity and
reduce off-target cleavage
 An increase in the protospacer adjacent motif
length is another strategy that is being used to
minimize off-target cleavage
26
El-Mounadi et al. (2019)

Future recommendations for the
CRISPR/Cas9 System
Future
recommendation
CRISPR/Cas9
System
Gene Knockout
CRISPR activation
Gene Knockdown
CRISPR + epigenetic modification
Gene Knock in
Small size new CRISPR
CRISPR system with no PAM site
Avoid off target effects
High-throughput
Base Editing 27

Case Study I
 First Report of CRISPR/Cas9 Mediated
DNA-Free Editing of 4CL and RVE7
Genes in Chickpea Protoplasts
 Author:- Sapna Badhan, Andrew S.
Ball and Nitin Mantri *
 Publish in:- International Journal of
molecular biology
 Published on:- 1 January 2021
 Objective:-study will help unravel the
role 4CL and RVE7 genes under
drought stress and understand the
complex drought stress mechanism
pathways.
28

Function of Gene:-
4CL- 4-coumarate ligase
 4-coumatrate: CoA ligase gene codes for coumarate ligase enzyme
which is well known for its role in the biosynthesis of secondary
plant metabolites during phenylpropanoid metabolism.
 phenylpropanoid enzyme is essential for the activation of the
hydroxycinnamic acids during lignin biosynthesis.
RVE7- Reveille 7
 RVE7 is a gene that encodes the transcription factor involved in
circadian rhythm and the opening of cotyledon mediated by
phytochrome A.
 RVE7 is active in controlling the circadian clock’s downstream
processes such as hypocotyl growth and flowering .
Badhan et al. (2021)29

Material Methodology:-
1. Chickpea Plant Material and Cas9 Protein
Commercial Kabuli chickpea plants were use.
The recombinant S. pyogenes Cas9 nuclease puriﬁed from an E.coli
strain expressing the nuclease was used in this study.
2. Target Site Selection and sgRNA Design
The sgRNA targets were designed using CHOPCHOP tool
drought tolerance associated genes (4CL and RVE7) were selected for
knockout based on their expression levels in ICC283 and ICC8261 under
drought stress
Gene Name
ICC8261
drought tolerant genotype
ICC283
drought sensitive genotype
RVE7
SAM-2.63,
FB-3.4,
FOF-1.29
YP-1.9
SAM-3.4
FB-3.02
FOF-5.9
YP-0
4CL FB-10.39 Not Differentially expressed data

3. In Vitro Cleavage Assay
4. Protoplast Isolation
Plant Media BioWORLD kit (Protoplast Isolation kit)
5. Protoplast Transformation with RNP Complex
 crRNA + transRNA + Cas9 = RNA cpmolex
 The sample treated only with the Cas9 enzyme was used as a negative
control.
6. DNA Extraction and PCR Ampliﬁcation of Target Region
Gene Name sgRNA sgRNA Sequence
4-coumarate-CoA ligase-like 1
NW_004516753.1_162798-165892
Length 3094
4CLsgRNA1
4CLsgRNA2
TATGTCACCGTCTAGTTCATTGG
GTTTAGGTTACCGAACGAAGAGG
REVEILLE 7-like
NW_004516329.1_420654-4253847
RVE7sgRNA1
RVE7sgRNA2
GTGGAGGATTGAATGTAAGACGG
AGTGTGCAGCTGATGTATCGAGG

Result:-
sgRNA Selection and Design
The sgRNA for the target location were designed using CHOPCHOP and veriﬁed by other
tools such as CCTop using the genome sequence for Kabuli chickpea.
Systematic design to show the location of sgRNA target sites in RVE7 and 4CL
nucleotide sequences.
(a) Systematic illustration of the nucleotide sequence of RVE7 gene locus.
(b) Systematic diagram of the nucleotide sequence of 4CL gene locus.
represent exons and connecting lines represent intron

sgRNA Selection and Design In Vitro Digestion Assay
The DNA of 5 kb PCR-amplified fragments for gene 4CL and RVE7 were treated with
preassembled RNP and in vitro cleavage assay was performed.
(a) sgRNA 1 for 4CL and RVE7.
 For in vitro cleavage assay non-treated samples were used as negative controls in
gel electrophoresis.
 expected band sizes for 4CL is 4931 and 1170;
 expected band sizes for RVE7 is 2298 and 3543

(a) sgRNA 2 for 4CL and RVE7.
 The digested samples for 4CL left side and RVE7 at right side.
 The expected band size for 4CL is 4469 and 1363.
 The expected band size for RVE-7 is 4477 and 1428

Conclusion:-
The results obtained from this study could help in developing new
traits and understanding the drought mechanism in chickpea
plants by knocking out the desired gene, followed by protoplast
regeneration or using the plant tissue for transformation.

Case Study II
 Programmed Editing of Rice (Oryza sativa
L.) OsSPL16 Gene Using CRISPR/Cas9
Improves Grain Yield by Modulating the
Expression of Pyruvate Enzymes and Cell
Cycle Proteins.
 Authors:- Babar Usman, Gul Nawaz,
Neng Zhao, Shanyue Liao, Baoxiang Qin,
Fang Liu, Yaoguang Liu and Rongbai Li
 Publish in :- International Journal of
Molecular Science
 Published:- 29 December 2020
 Objective:-
Improve Grain Yield by Modulating
the Expression of Pyruvate Enzymes
and Cell Cycle Proteins with the
addition of OsSPL16 Gene.
35
Usman et al. (2020)

OsSPL16 Gene
 OsSPL16 gene encodes a promoter binding protein that promotes cell
division and increases GWD.
 Loss-of-function mutations of OsSPL16 confer slender grain type and
better quality of appearance in Basmati rice
Material Methodology:-
1. Material Used and Experimental Conditions
 The Indica rice variety VP4892 was selected.
 The CRISPR/Cas9 intermediate vector pYLCRISPR/Cas9pubiH and
gRNA promoters (U6a and U6b) used in this study.
Usman et al. (2020)36

2. Target Site Selection and Vector Construction
 The OsSPL16 gene is located on chromosome 8,
 The OsSPL16 gene has a total length of 5032 bp.
 the amplification of the OsSPL16 gene was performed for VP4892 variety
by using specific primers (GW8F/R).
 two 20 bp long sgRNAs sequences followed by PAM were designed
 We selected five potential off-targets containing at least two nucleotide
mismatches for each target to analyze off-target effects

Construction of vector and Rice Transformation
 Adapter primersWx-U6-F/Wx-U6-R were use to construct the ligation reaction of
the sgRNA.
 The ligation product was used as a template for PCR amplification
 The expression cassette was transformed into A. tumefaciens EHA105 by
electroporation and rice transformation was achieved .
Genotyping, Phenotypic and Screening of T-DNA-Free Plants
 The target sites of the T0,T1 and T2 generations of the genetic transformation
material were sequenced and analyzed.
 The sequencing ﬁles were processed using the DSDecode M tool.
 The agronomic traits such as Plant height, number of panicle, Panicle length,
thousand grain weight, gran weight etc. mutant lines wee recorded in T0, T1 and
T2 generations.
Protein Preparation, Labeling, and Fractionation
Proteomic data analysis
 Proteome Discoverer 1.2 software is use.
Data Analysis
 Agronomic data were analyzed with SPSS 20.0 software, using Student’s t-test
 The graphs for agronomic data and proteomic data were developed by Graph Pad
Prism

Result:-
Validation of Targets Assembly and Genotyping of Mutant Plants
The amplified product was mixed and purified by TaKaRa MiniBEST Purification Kit
Ver.4.0
The purified product was sequenced using specific primers (SPL1/SPR; Table S1).
the constructed vector is suitable for the next step of Agrobacterium Mediated genetic
transformation of rice

Genotyping, and Protein Modeling
• 50 calli were treated with transformed A. tumefaciens and 12 rice plantlets were
obtained.
• The sequencing results displayed that 9 independent mutant lines showed
mutations in target sites, representing an editing efficiency of 75%.
First target
• There were 4 mono-allelic heterozygous, 2 bi-allelic heterozygous, 3
homozygous, and 3 Wild Type plantlets.
• GXU52-6 6 bp and GXU52-8 9 bp ) presented homozygous mutation
Second Target
• 3 monoallelic heterozygous, 1 bi-allelic heterozygous, 4 homozygous, and 4
WT plantlets at the second target.
• (GXU52-6 7 bp and GXU52-8 3 bp ) presented homozygous mutations
The predicted off-target regions were successfully amplified and there were no off-target
mutations found in selected five loci against both targets.

Agronomic Performance
We selected GXU52-3, GXU52-6, and GXU52-8 mutant lines for the investigations of
agronomic traits.
Generation Genotype Grain width 1000 seed wt. Yield
T0
WT 2.9 ± 0.2 30.3 ± 1.3 27.5 ± 2.9
GXU52-3 3.9 ± 0.2 43.9 ± 1.4 41.7 ± 1.6
GXU52-6 3. ± 0.1 44.5 ± 1.2 42.9 ± 2.2
GXU52-8 3.9 ± 0.3 44.6 ± 1.5 42.5 ± 1.3
T1
WT 2.9 ± 0.1 29.8 ± 1.4 28.2 ± 2.3
GXU52-3-1 3.8 ± 0.3 44.8 ± 1.5 42.1 ± 1.3
GXU52-6-1 3.9 ± 0.2 44.6 ± 1.3 41.9 ± 3.2
GXU52-8-1 3.9 ± 0.2 43.9 ± 1.2 42.3 ± 1.6
T2
WT 2.8 ± 0.3 31.1 ± 1.1 28.9 ± 2.5
GXU52-3-2 3.9 ± 0.2 44.9 ± 1.3 41.9 ± 2.6
GXU52-6-2 3.8 ± 0.1 44.7 ± 1.6 42.8 ± 2.1
GXU52-8-2 3.7 ± 0.3 44.7 ± 1.4 42.4 ± 1.8 41

(A) and grain phenotype (B) of wild type (WT) and mutant lines.
Seeds were randomly collected from GXU52-3, GXU52-6, and GXU52-8 mutant lines
for phenotyping.

Conclusion
 The targeted multiplex genome editing facilitated the
identification of some candidate proteins and biological pathways
that may involve in rice grain development.
 The targeted genome editing also facilitated a path way level study
for engineered rice mutants with enhanced grain yield.
 The OsSPL16 mutants laid an imperative material foundation for
additional application in stable and high yield breeding of rice.
43
Usman et al. (2020)

Case Study-III
 Engineering of CRISPR/Cas9-mediated
potyvirus resistance intransgene-free
Arabidopsis plants
 Authors:- Douglas E. Pyott, Emma
Sheehan And Attila Molnar
 Publish in :- MOLECULAR PLANT
PATHOLOGY
 Published:- 2016
 Objective:- potyvirus resistance
intransgene-free Arabidopsis plants
44

Materials and methods
 Plant growth conditions
 Guide RNA design and cloning
 Plant transformation
 BASTA selection
 PCR conditions
 T7 endonuclease assay
 Sanger sequencing
 Viral inoculations
 Viral GFP imaging and RT-PCR/quantitative RT-PCR
45
Douglas et al. (2016)

As the mutations in the T1
generation occurred in somatic
cells, and so were not
heritable, and different
mutations were recovered in
the T2 generation because of
independent editing events in
the germline of T1.
Non-transgenic T2 plants,
which were homozygous for
either the mutated or wild-type
eIF(iso)4e alleles, were used
to produce T3 populations,
which were then tested for
viral resistance.
46

47
polymerase chain reaction (PCR) was used to confirm the
presence/absence of the Cas9 transgene, using the constitutively
expressed house-keeping gene EF1a as a loading control.
A Cas9 transformant (T1 generation) and a non-transformed
wild-type plant were used as positive and negative controls for Cas9
amplification, respectively.
Samples #41–#49 are a representative selection of T2 progeny from
T1 plant number 1
Candidates #44 and #45 represent two of a total of 55 candidates
lacking the Cas9 transgene, which were selected by this method.

48
Summary of CRISPR/Cas9-
induced eIF(iso)4E mutations.
(A)DNA sequence alignments for
the four homozygous eIF(iso)4E
mutants (#44, #65, #68, #98)
identified in the T2 generation,
together with a wild-type (WT)
control.
Lines #65, #68 and #98 exhibit
single-nucleotide insertions,
whereas line #44 has a single-
nucleotide deletion.
(B) Predicted amino acid sequence
alignments for the four
homozygous mutants and the
wild-type consensus. Each of the
mutant alleles codes for severely
truncated and disrupted proteins

49
(A) Representative photographs of TuMV-
GFP virus-infected plants imaged
under UV light at 7 days post-infection.
 A transposon-induced eIF(iso)4E
mutant (Tn) was used as a resistant
control.
(B) RT-PCR to detect the presence of
TuMV-GFP in leaves for each genotype.
• Amplicons of the TuMV coat
protein region (537 bp) and the
house-keeping gene EF1a (418 bp)
were PCR amplified separately
from the same cDNA,
• TuMV-specific amplicons are
clearly visible in each of the wild-
type (WT) samples, but completely
absent from any of the eIF(iso)4E
mutant samples.
(C) Quantitative RTPCRs were performed
with cDNA from a healthy plant (H) and
water (W) as negative controls (NC). Error
bars show the standard error of the mean
(SEM) of three biological replicates.

 Back-inoculations of Nicotiana benthamiana plants using sap from TuMV-GFP
inoculated Arabidopsis.
 Sap was prepared by pooling 20 systemic leaves from TuMV-GFP-inoculated
Arabidopsis.
 Each quadrant shows an inoculated leaf (I) and systemic tissue (S) for two
replicate plants imaged under UV light.
50
Thomas et al. (2016)

51
Box plots of dry weights (A) and flowering times (B) for the
CRISPR/Cas9-edited eIF(iso)4E mutants (lines #44, #65, #68
and #98) alongside a wild-type (WT) plant (#105)

Conclusion
 In this study, we have showcased the utility of CRISPR/Cas9
technology for the generation of novel genetic resistance to TuMV
in Arabidopsis by the deletion of a host factor [eIF(iso)4E] which
is strictly required for viral survival.
52
Thomas et al. (2016)

Crop Gene Gene Function
Mutation
type
Editing
efficiency
(%)
Reason for
transformatio
n
Method of
Cas9 system
delivery
Reference
Rice (O.
sativa L.)
TMs5
TMS5 is
thermosensitive genic
male sterility
(TGMS) gene in
China which encodes
the endonuclease
RNase ZS1 in AnS-1
Single
nucleotide
insertions,
deletion and
substitutions
46.2 to
88.2
To develop
commercial
TGMS rice
lines
Agrobacteri
m mediated
delivery
method
.Zhou, H. et
al. (2016)
ALS
Encodes acetolactate
synthase, which is
involved in the
biosynthesis of the
branched amino acid
Point
mutations
Knock-in and
resistant
against
sulfonylurea
herbicides
Agrobacteri
m mediated
transformati
on method
Sun, Y. et al.
(2016)
Wheat
(T.
aestivum
L.)
TaMLO
homologs
Involved to inhibit
resistance pathway to
powdery mildew
Insertion and
deletion
mutations
23–38
To increase
resistance
against
powdery
mildew in
wheat
Particle
bombardmen
t method
Wang, Y. et
al. (2014)
TaGW2
TaGW2 gene plays a
vital role in grain
weight control
Insertion and
deletion
mutations
41.2
For efficient
and specific
genome editing
Cas9-
RNPmediate
d GE method
Liang, Z. et
al. (2017)
Upland
cotton
(G.
hirsutum
L.)
GhCLA1
(Chloroplast
s alterados
1)
Nucleotide
insertion and
substitution
47.6–81.8
For targeted
mutagenesis of
cotton genome
Agrobacteriu
m mediated
transformati
on method
Chen, X. et
al. (2017)
53

Crop Gene Gene Function Mutation type
Editing
efficiency
(%)
Reason for
transformation
Method of
Cas9 system
delivery
Reference
Upland
cotton (G.
hirsutum
L.)
GhVP (vacuolar
H+ -
pyrophosphatase)
Nucleotide
deletion and
substitution
47.6–81.8
For targeted
mutagenesis of
cotton genome
Agrobacteriu
m mediated
transformati
on method
Chen, X. et
al. (2017)
An endogenous
gene GhCLA1
and DsRed2
(Discosoma red
fluorescent
protein2)
AtCLA1 is involved
in the development
of chloroplast.
DsRed2 protein is
utilized as a reporter
due to its different
benefits over other
report proteins
Nucleotide
insertions and
deletions
66.7–100
For targeted
mutagenesis of
cotton genome
Agrobacteriu
m mediated
transformati
on and
somatic
embryogene
sis method
Wang, P. et
al. (2018)
GhMYB 25-like
GhMYB25-like is
involved in the
development of
cotton fiber
Nucleotide
insertion and
deletion
mutations
100 and
98.8
For efficient and
specific genome
editing
Agrobacteriu
m mediated
transformati
on and
somatic
embryogene
sis method
Li, C. et al.
(2017)
Maize (Z.
mays L.)
ZmAgo18a and
ZmAgo18b
(Argonaute 18)
and
Dihydroflavonol
4- reductase or
anthocyaninless
(a1 and a4)
Involved in the
biosynthesis of 24-
nt phasiRNA in
anthers
Showed monoor
diallelic
mutations of one
locus and
various allelic
variations of two
loci
70
For mutagenesis
frequency and
heritability
Agrobacteriu
m mediated
transformati
on method
Char, S.N. et
al. (2017)
54

Mutation
type
Editing
efficiency
(%)
Reason for
transformati
on
Method of
Cas9 system
delivery
Reference
Soybean
(G. max
L. Merr.)
GmPPD1
and
GmPPD2
PPD protein is
involved in the
transcriptional
regulation of cell
division in Arabidopsis
Heterozygou
s and
chimeric
mutations
68 in
GmPPD1
and 88 in
GmPPD2
Inheritable
sitedirected
mutagenesis
Agrobacteriu
m mediated
transformatio
n method
Kanazashi,
Y. et al.
(2018)
GmFT2a
GmFT2a is an
integrator in the
photoperiod flowering
pathway
Site-directed
and insertion
and deletion
(indels)
mutations
48, 53 and
37
To induce
targeted
mutagenesis
of GmFT2a
Agrobacteriu
m
tumefaciensm
ediated
transformatio
n method
Cai, Y. et al.
(2018)
Tomato
(S.
lycopersi
cum L.)
SlPDS
(phytoene
desaturase)
and SlPIF4
(phytochrom
e interacting
factor)
SlPDS are involved in
carotenoid
biosynthesis. The
SlPIF4 is a
homologous gene of
Arabidopsis PIF4,
which belongs to the
basic helix-loophelix
multigene family
Insertions
and
deletions
(indels)
83.56
For targeted
mutagenesis
in tomato
plants
Agrobacteriu
m
tumefaciensm
ediated
transformatio
n method
Pan, C. et
al. (2016)
SlIAA9
(auxininduce
d 9)
SlIAA9 is a key gene
controlling
parthenocarpy
Insertions
and
deletions
(indels)
100
To generate
parthenocarpi
c tomato
plants
Agrobacteriu
m
tumefaciensm
ediated
transformatio
n method
Ueta, R. et
al. (2017)
55

Crop Gene Gene Function Mutation type
Editing
efficiency
(%)
Reason for
transformation
Method of
Cas9 system
delivery
Reference
Barley
(Hordeum
vulgare
L.)
HvPM19
HvPM19 encodes an
ABA-inducible plasma
membrane protein that is
involved in the positive
regulation of grain
dormancy in wheat
Insertion and
deletion
(indels)
mutations
23 and 10
To induce
targeted
mutagenesis of
barley genes
Agrobacteriu
m mediated
transformatio
n method
Lawrenson,
T. et al.
(2015)
Sorghum
(S. bicolor
L.
Moench)
Whole k1C
gene family
Kafirins are proteins that
are used as storage in
Sorghum grains and form
protein bodies with poor
digestibility
Insertion and
deletion
(indels)
mutations
92.4
To create kafirin
variants for the
improvement of
protein
digestibility and
quality
A.
tumefaciensm
ediated
transformatio
n method
Li, A. et al.
(2018)
Rapeseed
(Brassica
napus L.)
RGAs, FULs,
DAs, and
A2.DA2
RGAs act as a master
repressor in gibberellic
signaling. The BnaFULs
are involved in the
regulation of silique
dehiscence during flower
development. The da2
and da1 are serving as
negative regulators of
organ size
Homozygotes,
bialleles, and
heterozygotes
65.3
To induce
targeted genome
modifications at
multiple loci
Agrobacteriu
m mediated
transformatio
n method
Yang, H. et
al. (2017)
Rapeseed
SPL3
homologous
gene copies
SPL3 is key floral
activator which acts
upstream of AP1 in
Arabidopsis
Insertion and
deletion
(indels)
mutations
96.8–100
To rapidly
generate and
identify
simultaneously
mutagenesis of
multiple gene
homologs
Li, C. et al.
(2018)
56

Mutation
type
Editing
efficiency
(%)
Reason for
transformat
ion
Method of
Cas9
system
delivery
Reference
Brassica
oleracea
BolC.GA4.
a
GA4 is involved in the
gibberellin
biosynthesis pathway
Insertion and
deletion
(indels)
mutations
10
To induce
targeted
mutagenesis
of B.
oleracea
genes
Agrobacter
ium
mediated
transformat
ion method
Lawrenson, T.
et al. (2015)
Potato
(Solanum
tuberosu
m)
GBSS
(granulebou
nd starch
synthase)
GBSS is responsible
for the synthesis of
amylose
Mutation is
alleles, small
insertions,
and/ or
deletions
(indels)
mutations
Up to 67
In order to
alter the
starch
quality
Transient
transfectio
n and
regeneratio
n from
isolated
protoplasts
Andersson, M.
et al. (2017)
Cucumber
(Cucumis
sativus
L.)
eIF4E
(eukaryotic
translation
initiation
factor 4E)
gene
eIF4E is a plant
cellular translation
factor which plays the
crucial role in the
Potyviridae life cycle
Small
deletions and
single
nucleotide
polymorphis
ms (SNPs)
In order to
enhance
tolerance
against the
virus in
cucumber
A.
tumefacien
smediated
transformat
ion method
.
Chandrasekara
n, J. et al.
(2016)
Watermel
on
ClPDS
(phytoene
desaturase)
ClPDS introduces
obvious albino
phenotype
Insertions or
deletions
100
To
effectively
create
knockout
mutations in
watermelon
Agrobacter
iummediat
ed
transformat
ion method
Tian, S. et al.
(2017)
57

Enhancing Abiotic Stress Tolerance In Plant
Crop Gene
Symbol
(gene/QTL)
Target trait References
Rice
calcium-dependent lipid binding annexing OsAnn3 Cold Shen et al. (2017)
Oryza sativa ethylene response factor (ERF) gene
(OsDERF1)
Oryza sativa photo-period sensitive male sterile
(OsPMS3)
OsDERF1,
OsPMS3,
OsEPSPS,
OsMSH1,
OsMYB5
Drought
Zhang et al.
(2014)
osmotic stress/ABA–activated protein kinase 2 OsSAPK2 Drought Lou et al. (2017)
NAM, ATAF and CUC (NAC) transcription factors OsNAC041 Salinity Bo et al. (2019)
Mitogen-activated protein kinase
OsMPK2,
OsDEP1
Yield under
stress
Shan et al. (2014)
Wheat Dehydration responsive element binding
TaDREB2,
TaERF3
Abiotic
stress
Kim et al. (2018)
Tomato
C-repeat-binding factor CBF1
Chilling
tolerance
Li et al. (2018)
Stable tomato non expresser of pathogenesis-related
gene 1
SlNPR1 Drought
Acetolactate synthase
SlALS1,
SlALS2
Herbicide
Veillet et al.
(2019a) 58

Conclusion
 CRISPR is the most powerful tool of
biotechnology.
 With the help of this technology we design the
crop as per our need by modifying the genome
of the crop.
59
Thank You………..

Literature Cited
 Andersson, M. et al. (2017) Efficient targeted multiallelic mutagenesis in tetraploidpotato (Solanum
tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep. 36,
117–128
 Anjanabha Bhattacharya Vilas Parkhi Bharat Char (2020) Editors CRISPR/Cas Genome Editing
book Springer
 Babar Usman 1 , Gul Nawaz 1 , Neng Zhao 1 , Shanyue Liao 1 , Baoxiang Qin 1 , Fang Liu 1 ,
Yaoguang Liu 2,* and Rongbai Li 1,* (2021) Programmed Editing of Rice (Oryza sativa L.)
OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of
Pyruvate Enzymes and Cell Cycle Proteins Int. J. Mol. Sci., 22, 249
 Bo W, Zhaohui Z, Huanhuan Z, Xia W, Binglin L, Lijia Y, Xiangyan H, Deshui Y, Xuelian Z,
Chunguo W (2019) Targeted mutagenesis of NAC transcription factor gene, OsNAC041,
leading to salt sensitivity in rice. Rice Sci 26:98–108
 Cai, Y. et al. (2018) CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time
in soya bean. Plant Biotechnol. J. 16, 176–185
 Chandrasekaran, J. et al. (2016) Development of broad virus resistance in non-transgenic
cucumber using CRISPR/Cas9 technology. Mol. Plant Pathol. 17, 1140–1153
 Char, S.N. et al. (2017) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency
targeted mutagenesis in maize. Plant Biotechnol. J. 15, 257–268
 Chen, X. et al. (2017) Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/
Cas9 system. Sci. Rep. 7, 44304
60

61
 Deepa Jaganathan*, Karthikeyan Ramasamy, Gothandapani Sellamuthu, Shilpha Jayabalan and
Gayatri Venkataraman* (2018) CRISPR for Crop Improvement: An Update Review. Front.
Plant Sci. 9:985.
 Hakim Manghwar,1,3 Keith Lindsey,2 Xianlong Zhang,1,3, * and Shuangxia Jin1,3, * (2019)
CRISPR/Cas System: Recent Advances and Future Prospects for Genome Editing Trends in
Plant Science, Vol. 24, No. 12 https://doi.org/10.1016/j.tplants.2019.09.006
 Kaoutar El-Mounadi 1 , María Luisa Morales-Floriano2,3 and Hernan Garcia-Ruiz 3* Principles,
Applications, and Biosafety of Plant Genome Editing Using CRISPR-Cas9(2020) Front.
Plant Sci. 11:56.
 Kah-Yung Bernard Leong, Yee-Han Chan, Wan Muhamad Asrul Nizam Wan Abdullah, Swee-Hua
Erin Lim and Kok-Song Lai(2018) The CRISPR/Cas9 System for Crop Improvement:
Progress and Prospects IntechOpen (http://creativecommons.org/licenses/by/3.0)
 Kanazashi, Y. et al. (2018) Simultaneous sitedirected mutagenesis of duplicated loci in soybean using
a single guide RNA. Plant Cell Rep. 37, 553–563
 Kim D, Alptekin B, Budak H (2018) CRISPR/Cas9 genome editing in wheat. Funct Integr
Genomics 18:31–41
 Lawrenson, T. et al. (2015) Induction of targeted, heritable mutations in barley and Brassica
oleracea using RNA-guided Cas9 nuclease. Genome Biol. 16, 258
 Liang, Z. et al. (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9
ribonucleoprotein complexes. Nat. Commun. 8, 14261
 Li R, Zhang L, Wang L, Chen L, Zhao R, Sheng J, Shen L (2018) Reduction of tomato-plant
chilling tolerance by CRISPR–Cas9-mediated SlCBF1 mutagenesis. J Agric Food Chem
66:9042–9051
 Li, C. et al. (2017) A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton
(Gossypium hirsutum L.). Sci. Rep. 7, 43902

62
 Li, A. et al. (2018) Editing of an alpha-kafirin gene family increases, digestibility and protein
quality in Sorghum. Plant Physiol. 177, 1425–1438
 Li, C. et al. (2018) An efficient CRISPR/Cas9 platform for rapidly generating simultaneous
mutagenesis of multiple gene homoeologs in allotetraploid oilseed rape. Front. Plant
Sci. 9, 442
 Lou D, Wang H, Liang G, Yu D (2017) OsSAPK2 confers abscisic acid sensitivity and
tolerance to drought stress in rice. Front Plant Sci 8:993
 Pan, C. et al. (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in
tomato plants in the first and later generations. Sci. Rep. 6, 24765
 Sapna Badhan, Andrew S. Ball and Nitin Mantri * (2021) First Report of CRISPR/Cas9
Mediated DNA-Free Editing of 4CL and RVE7 Genes in Chickpea Protoplasts Int. J.
Mol. Sci., 22, 396.
 Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas
system. Nat Protoc 9:2395
 Shen C, Que Z, Xia Y, Tang N, Li D, He R, Cao M (2017) Knock out of the annexin gene
OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice.
J Plant Biol 60:539–547
 Sun, Y. et al. (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated
homologous recombination of acetolactate synthase. Mol. Plant 9, 628–631
 Tian, S. et al. (2017) Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell
Rep. 36, 399–406
 Thomas B Jacobs1,3,5*, Peter R LaFayette2,3, Robert J Schmitz4 and Wayne A Parrott1,2,3
(2015) Targeted genome modifications in soybean with CRISPR/Cas9 BMC
Biotechnology15:16 DOI 10.1186/s12896-015-0131-2

63
 Ueta, R. et al. (2017) Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Sci.
Rep. 7, 507
 Veillet F, Chauvin L, Kermarrec M-P, Sevestre F, Merrer M, Terret Z, Szydlowski N, Devaux P,
Gallois J-L, Chauvin J-E (2019a) The Solanum tuberosum GBSSI gene: a target for
assessing gene and base editing in tetraploid potato. Plant Cell Rep 1–16
 Wang, P. et al. (2018) High efficient multisites genome editing in allotetraploid cotton (Gossypium
hirsutum) using CRISPR/Cas9 system. Plant Biotechnol J. 16, 137–150
 Wang, Y. et al. (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat
confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951
 Yang, H. et al. (2017) CRISPR/Cas9-mediated genome editing efficiently creates specific
mutations at multiple loci using one sgRNA in Brassica napus. Sci. Rep. 7, 7489
 Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N (2014) The
CRISPR/C as9 system produces specific and homozygous targeted gene editing in rice in
one generation. Plant Biotechnol J 12:797–807
 Zhou, H. et al. (2016) Development of commercial thermo-sensitive genic male sterile rice
accelerates hybrid rice breeding using the CRISPR/Cas9- mediated TMS5 editing system.
Sci. Rep. 6, 37395

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

More Related Content

What's hot (20)

Similar to CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) (20)

More from Akshay Deshmukh (7)

Recently uploaded (20)

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

Editor's Notes