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© Springer Nature Singapore Pte Ltd. 2020
A. Rakshit et al. (eds.), New Frontiers in Stress Management for Durable
Agriculture, https://doi.org/10.1007/978-981-15-1322-0_23
CRISPR/Cas9-Edited Rice: ANew Frontier
forSustainable Agriculture
SahilMehta, ShambhuKrishanLal,
KuleshwarPrasadSahu, AjayKumarVenkatapuram,
MukeshKumar, VijaySheri, PanditiVarakumar,
ChandrapalVishwakarma, RenuYadav, M.RizwanJameel,
MirajAli, V.MohanM.Achary, andMalireddyK.Reddy
Abstract
With the exponential increase in the world’s human population, improving agri-
cultural productivity is among the top of the researchers’ agendas till the 2050
deadline. One of the potential solutions to this global issue is genome editing
because of the precision, fastness, and probably low cost involved compared to
other traditional methods. It is in the spotlight especially from the last decade due
to the discovery of sequence-specic-based nuclease technology including
CRISPR/Cas9 tool. Initially, this tool was applied only in protoplasts and calli.
However, due to the modications in vectors, Cas9 variants, cassettes, cloning
systems, multiplexing, and delivery methods, this platform has revolutionized
the plant science eld. It has been exploited in such a manner that about 16 crop
plants have been already edited in the last few years. Out of all crops, most of the
editing has been done in the case of rice (Oryza sativa L., Family: Poaceae), a
cereal staple food. Therefore, in the current chapter, we have highlighted about
S. Mehta · A. K. Venkatapuram · V. Sheri · P. Varakumar · R. Yadav · M. Ali · V. M. M. Achary ·
M. K. Reddy
Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology,
New Delhi, India
S. K. Lal (*)
Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology,
New Delhi, India
K. P. Sahu · M. Kumar
Division of Plant Pathology, ICAR-Indian Agriculture Research Institute, New Delhi, India
C. Vishwakarma
Division of Plant Physiology, ICAR-Indian Agriculture Research Institute, New Delhi, India
M. R. Jameel
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia University,
New Delhi, India
23
428
the CRISPR/Cas9-edited rice for agronomic traits, stress tolerance/resistance,
and biofortication. Additionally, we have presented an overview of various
tools, databases, and commercial service providers devoted solely to CRISPR/
Cas9 genome-editing technology.
Keywords
Plants · Agriculture · Yield · CRISPR/Cas · Online resources · Future crops
23.1 Introduction
In today’s world, the human population is increasing exponentially and is expected
to cross the whopping mark of 9.7 billion by the year 2050 (Valin etal. 2014; Baltes
et al. 2017; Figueroa 2019). Furthermore, the whole scenario is expected to be
affected greatly by the need to generate more space, reduce the overexploitation of
natural resources, and tackle the uncertainties of climatic conditions and global
warming (Cazzolla Gatti 2016; Oldeman et al. 2017; Morton et al. 2017;
Subramanian 2018; Philander 2018; Pradinaud etal. 2019). In addition to this chal-
lenges, international food security, ghting chronic malnourishment, increasing
awareness, and interest for healthier functional foods are at the top of the agendas
(Siro et al. 2008; Abuajah et al. 2015; Martirosyan and Singh 2015; Atkins and
Bowler 2016; Baltes etal. 2017; Pratim Roy 2019).
As our contemporary agricultural lands are degrading, it necessitates to re-think
about the current agricultural practices, generation of elite varieties as well as ef-
cient distribution of food (Wingeyer etal. 2015; Morton etal. 2017; Glenn et al.
2017; Banasik etal. 2017; Zhang etal. 2018; Dillard 2019). Solutions to all these
challenges are unlikely to come from cross-breeding and mutation breeding (Kantar
etal. 2019; Belkhodja 2018; Chen etal. 2019; Kleter etal. 2019; Mehta etal. 2019a;
Singh etal. 2019; Rahman etal. 2019). Cross-breeding takes a large span of years
to introduce desirable alleles (Darwin 2010; Scheben etal. 2017). Furthermore, this
is limited by greatly reduced genetic variability. On the other hand, mutation breed-
ing usually employs agents like ethyl methanesulfonate (EMS) and gamma rays to
expand genetic variation by introducing random mutations (Bado etal. 2015, 2017;
Pacher and Puchta 2017; Xuan etal. 2019). However, it is restricted by the large-
scale mutant screening, high randomness, low efciency, and stochastic nature.
Furthermore, these approaches cannot keep pace with the whopping demand for
increased crop production.
As a result, one of the potent approaches that can withstand the increasing crop
productivity is genetic engineering (Marco etal. 2015; Baret and Vanloqueren 2017;
Knott and Doudna 2018). It has been the spotlight around the globe to create new
crop varieties (Sticklen 2008; Marco et al. 2015; Azadi et al. 2016; Arzani and
Ashraf 2016; Kumari etal. 2018; Waltz 2018; Banerjee and Roychoudhury 2019;
Zhang etal. 2019). Generally, it is dened as the targeted modication of DNA of
any living organism belonging to any kingdom of classication using various tools
S. Mehta et al.
429
(Baltes etal. 2017). In accordance with the current and future scenario challenges,
it easily addresses questions like (1) which traits need to be introduced, (2) which
crops need to be focused on, (3) which DNA modications must be done to generate
the desired traits in the selected crops, (4) how to introduce these DNA modica-
tions in the crop’s genome, (5) how to overcome the bottlenecks of existing tools for
crop improvement particularly, and (6) how to shift the agendas in accordance with
the changing challenges. Due to the wide-ranging use, the enormous number of
application falls under the big umbrella of genome engineering. As a result there is
a wide range of potential products that could address food security/quality issues
(Hsu etal. 2014; Wu etal. 2016; Nielsen and Keasling 2016; Khalid etal. 2017;
Knott and Doudna 2018; Shigaki 2018; Waltz 2018; Pray etal. 2018; Merga etal.
2019; Zhang 2019).
Nonetheless, one of the signicant tools that has been used enormously in agri-
culture is genome editing (Upadhyay etal. 2013; Laible etal. 2015; Alagoz etal.
2016; Ricroch etal. 2017; Gao 2018; Eş etal. 2019; Lassoued etal. 2019; Yin and
Qiu 2019). This is truly reected in numerous improved cultivars which have
emerged within the last decade (Laible etal. 2015; Alagoz et al. 2016; Yin etal.
2017; Gao 2018; Yin and Qiu 2019). Here, we have highlighted different types of
genome-editing tools for plants. Additionally, we have focused on the CRISPR/
Cas9-edited rice for various traits and the current limitations and challenges within
this eld.
23.2 Genome-Editing Techniques forPlants
Perhaps the availability of numerous tools for DNA/RNA modications, the
sequence-specic nucleases have been the spotlight for the entire last decade
(Porteus and Carroll 2005; Wright etal. 2005; Christian etal. 2010; Voytas 2013;
Sprink etal. 2015; Zischewski etal. 2017; Waltz 2018; Novak 2019). These nucle-
ases introduce targeted DNA double-strand breaks (DSBs) which are repaired by
the cells itself by two evolved pathways, i.e., homologous recombination (HR) and
nonhomologous end joining (NHEJ) (Puchta and Fauser 2014). However, in com-
parison, NHEJ is naturally an error-prone pathway which frequently results in small
indels at the repair sites. Therefore, the researchers utilize this for targeted mutagen-
esis at a locus of interest. This NHEJ pathway exists in somatic, meiotic, and mitotic
cells throughout the cell cycle, whereas HR-mediated repair pathway occurs only
within the G2 and S phases of cells having mitotic activity (Huang and Puchta 2019;
Jun etal. 2019).
In the present scenario, mostly genome editing is done by multiple technologies
like meganucleases (Certo etal. 2012; Daboussi etal. 2015; Youssef etal. 2018),
zinc-nger nucleases (ZFNs) (Porteus and Carroll 2005; Wright etal. 2005; Bilichak
and Eudes 2016; Novak 2019), TALENs (Christian etal. 2010; Bilichak and Eudes
2016; Hensel and Kumlehn 2019), and CRISPR/Cas9 systems in plants (Bilichak
and Eudes 2016; Knott and Doudna 2018; Waltz 2018; Huang and Puchta 2019). A
detailed comparison of all these editing technologies is tabulated in Table23.1. For
more detailed information, the readers can look for publications by Gaj etal. (2013),
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
430
Table 23.1 A tabular comparison of major genome-editing technologies in plants
S.No. Attributes ZFNs TALENs CRISPR/Cas9
1 Cleavage type Protein-dependent Protein-dependent RNA-dependent
2 Size Signicantly
smaller than Cas9
(+)
Comparatively larger
than ZFNs (++)
Signicantly larger
than both ZFNs and
TALENs (+++)
3 Components Zinc-nger
domains,
nonspecic FokI
nuclease domain
TALE DNA-binding
domains, nonspecic
FokI nuclease
domain
Cas9 protein, crRNAs
4 Catalytic
domain(s)
FokI endonuclease
domain
FokI endonuclease
domain
HNH, RUVC
5 Structural
components
(dimeric/
monomeric)
Dimeric Dimeric Monomeric
6 Target sequence
length
18–36 24–59 20–22
7 gRNA
production
required
No No Yes
8 Cloning required Yes Ye s No
9 Protein
engineering steps
needed
Yes Yes No
10 Mode of action Induce DSBs in
target DNA
Induce DSBs in
target DNA
Induce DSBs or
single-strand DNA
nicks in target DNA
11 Restriction target
site
High G 5′T and 3′APAM sequence
12 Level of target
recognition
efciency
High High Very high
13 Targeting Poor Good Very good
14 Mutation rate
level
High Low Very low
15 Off-target effects Yes Yes Yes, but can be
minimized by the
selection of unique
crRNA sequence
16 Cleavage of
methylated DNA
possible
No No Yes, but it will be
explored more
17 Multiplexing
enabled
Highly difcult Highly difcult Yes
18 Labor
intensiveness in
experiment setup
Yes Yes No
(continued)
S. Mehta et al.
431
Puchta and Fauser (2014), Sprink etal. (2015), Bilichak and Eudes (2016), Noman
etal. (2016), Baltes and Voytas (2015), Baltes etal. (2017), Malzahn etal. (2017),
Kamburova etal. (2017), Lino etal. (2018), Shah etal. (2018), and Novak (2019).
23.3 CRISPR/Cas9 System forFathomless Genetic
Engineering
Currently, the most popular genetic cargo technology is CRISPR/Cas9 (Shan etal.
2013; Belhaj etal. 2013; Miao etal. 2013). This system has truly revolutionized the
plant science research (Bilichak and Eudes 2016; Knott and Doudna 2018; Waltz
2018; Huang and Puchta 2019). As a result, various articles have been published
throughout the last few years (Belhaj etal. 2013; Shan etal. 2014; Gao etal. 2015;
Bilichak and Eudes 2016; Liu etal. 2017a, b; Liang etal. 2017; Knott and Doudna
2018; Butt etal. 2018; Abbott and Qi 2018; Huang and Puchta 2019). This is even
supported by the fact that the keyword “CRISPR/Cas” in the paper title fetched
about 5610 publications in Google Scholar (https://scholar.google.co.in/).
This CRISPR/Cas9 tool is popular due to the advantages such as simplicity, easy
design, and easiness in delivery (Upadhyay etal. 2013; Baltes etal. 2017; Langner
etal. 2018; Soda etal. 2018; Chen etal. 2019). CRISPR/Cas9 stand for clustered
regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated
endonuclease 9 (Cas9) (Shan etal. 2013; Baltes etal. 2017). Both are integral com-
ponents of the adaptive immunity system present within bacteria and archaea for
protection against bacteriophages (Horvath and Barrangou 2010; Bondy-Denomy
etal. 2013; Sampson etal. 2013; Shan etal. 2014). Based on this immunity mecha-
nism, the CRISPR/Cas9 plant transformation vectors have been designed which
carries guide RNA (gRNA) and Cas9 (Cong etal. 2013). In the initial days, it was
applied in protoplast, calli, germ cells, and somatic cells (Shan etal. 2013; Feng
etal. 2013, 2014a, b; Shen etal. 2014; Xing etal. 2014; Yin etal. 2015; Bhowmik
Table 23.1 (continued)
S.No. Attributes ZFNs TALENs CRISPR/Cas9
19 Possible to
generate
large-scale
libraries
No Yes, but it is highly
challenging
Yes
20 Design
feasibility
Difcult Difcult Easy
21 Technology cost Very high
(£1000–£3000)
High (£40–£350) Comparatively low
(£30–£300)
22 First report in
plants
Durai etal. (2005),
Lloyd etal. (2005)
Christian etal.
(2010)
Feng etal. (2013),
Shan etal. (2013),
Miao etal. (2013)
23 First report in
rice
Kim etal. (2012) Li etal. (2012) Jiang etal. (2013),
Shan etal. (2013),
Miao etal. (2013)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
432
et al. 2018). Until now, various modications have been done in CRISPR/Cas9
plant vectors (Shen etal. 2014; Ma et al. 2015; Mikami et al. 2015a, b; Osakabe
etal. 2016; Tsutsui and Higashiyama 2017; Wang etal. 2018; Wu etal. 2018; Mahas
etal. 2019). This is even supported by the fact that the optimized protocols are avail-
able for many plant species (Miao etal. 2013; Xing etal. 2014; Lowder etal. 2015;
Char etal. 2017; Bhowmik etal. 2018; Osakabe et al. 2018; Li and Zhang 2019).
Additionally, there is a plethora of available tools and databases devoted tothe vari-
ous omics technologies (Anamika etal. 2019) as well as CRISPR/Cas9 (Tables 23.2
and 23.3). Furthermore, there are many commercial service providers in the market-
place which provide many services and products related to the CRISPR/Cas9 tech-
nology (Table23.4).
Furthermore, this CRISPR/Cas9-mediated genome-editing tool has been suc-
cessfully implied in various plants (Cong etal. 2013; Upadhyay etal. 2013; Feng
etal. 2014a, b; Shan etal. 2014; Svitashev etal. 2015; Malnoy etal. 2016; Alagoz
etal. 2016; Liu etal. 2017a, b; Soda etal. 2018). For more detailed information, the
researchers are advised to look for publication from the Korotkova and group
(Korotkova etal. 2017, 2019).
Recently, Korotkova and colleagues published a cataloging article entitled
“Current achievements in modifying crop genes using CRISPR/Cas system”
(Korotkova et al. 2019). They studied all the published research articles on crop
genome modications from the Scopus database. In their article, they reported
CRISPR/Cas-based genome-editing technology has been applied largely to the rice.
The probable reason is being an established model plant which simultaneously
counted as the highly valued cash crop worldwide (Khush 2005). This is even
boosted by the availability of the rice genome sequence, sequence maps and multi-
ple databases (Goff etal. 2002; Yu etal. 2002; Project, I.R.G.S. and Sasaki 2005;
Smita etal. 2011; Zhao etal. 2014; Copetti etal. 2015; Zhang etal. 2016; Crossa
et al. 2017). This is even supported by the surge in the number of publications
related to the CRISPR/Cas9 (Fig. 23.1). Figure 23.2 highlights the key develop-
ments in the eld of CRISPR/Cas9 technology for rice.
Typically, the CRISPR/Cas9 system success in rice relies mostly on two factors:
(1) type of plant transformation vector and (2) the used delivery system. In general,
the vectors carry essentially Cas9 (a endonuclease/nickase), T-DNA border region,
selectable marker genes (plant and bacterial), ori site, and gRNA(s) (Alok etal.
2018) depending on the type of strategy-employed binary system, co- transformation,
and/or multiplexing (Fig.23.3). For more detailed information about the CRISPR/
Cas9 vector components, the readers can look for publication by Alok etal. (2018).
Similarly, the CRISPR/Cas9-editing reagents (DNA/RNa,RNPs) are delivered
into plant cells by particle bombardment (Shan etal. 2014; Sun etal. 2016; Li etal.
2016a, b, c, 2019), Agrobacterium-mediated transformation (Shan etal. 2013, 2014;
Xu etal. 2014; Hu etal. 2016; Lu and Zhu 2017; Wang etal. 2019), or protoplast
transfection (Xie and Yang 2013; Tang et al. 2019; Lin et al. 2018). The overall
workow for rice genome editing using CRISPR/Cas9 is depicted in Fig.23.3.
S. Mehta et al.
433
Table 23.2 Tabular account of available CRISPR/Cas9 tools in plants
S.No. Tool Specication Website URL address Provider References
1 CRISPR.mit Tool to facilitate the design of gRNAs http://crispr.mit.edu/ Zhang Lab Hsu etal. (2013)
2 sgRNA designer Online tool for effective sgRNA
designing
http://portals.broadinstitute.
org/gpp/public/analysis-
tools/sgrna-design
Broad Institute Doench etal.
(2014)
3 E-CRISP Web application to design gRNA
sequences
http://www.ecrisp.org/
ECRISP/
German Cancer Research Center Heigwer etal.
(2014)
4 CRISPRseek Part of R programming package for
designing gRNAs
http://www.bioconductor.
org/packages/release/bioc/
html/CRISP Rseek.html
Bioconductor Zhu etal. (2014)
5 Cas-OFFinder Algorithm for identifying potential
off-target sites in a genome
http://www.rgenome.net/
cas-ofnder/
Seoul National University Bae etal. (2014)
6 CHOPCHOP Online tool for predicting off-target
binding of sgRNAs
http://chopchop.rc.fas.
harvard.edu/
Harvard University Montague etal.
(2014)
7 CRISPRscan sgRNA-scoring algorithm that
effectively captures the activity of
CRISPR/Cas9 invivo
http://www.crisprscan.org/ Giraldez Lab (Yale University) Moreno-Mateos
etal. (2015)
8 CRISPRdirect Web server for selecting rational
CRISPR/Cas targets based on input
sequence
http://crispr.dbcls.jp/ Database Center for Life Science Naito etal.
(2014)
9 PROTOSPACER Web interface for nding, evaluating and
sharing Cas9 guide-RNA designs
http://www.protospacer.
com/
BIHP-Institute Pasteur (France) MacPherson and
Scherf (2015)
10 sgRNA Scorer 1.0 In vivo library methodology to assess
sgRNA activity
http://crispr.med.harvard.
edu/sgRNAScorerV1/
Wyss Institute for Biologically
Inspired Engineering at Harvard
Chari etal.
(2015)
11 CRISPR
Multi-Targeter
Online tool to nd sgRNA targets http://www.multicrispr.net/ IWK Health Centre and
Dalhousie University
Prykhozhij etal.
(2015)
(continued)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
434
Table 23.2 (continued)
S.No. Tool Specication Website URL address Provider References
12 Off-Spotter An algorithm to assist in designing
optimal gRNAs
http://cm.jefferson.edu/
Off-Spotter/
Thomas Jefferson University Pliatsika and
Rigoutsos
(2015)
13 WU-CRISPR Web tool for the genome-wide design of
sgRNAs
http://crispr.wustl.edu Xiaowei Wang Lab Wong etal.
(2015)
14 Breaking-Cas Web tool to facilitate the design of guide
RNA for CRISPR/Cas technique
http://bioinfogp.cnb.csic.es/
tools/breakingcas
Spanish National Center for
Biotechnology
Oliveros etal.
(2016)
15 CHOPCHOP v2 An updated version of CHOPCHOP
which improves the targeting power,
usability, and efciency of CHOPCHOP
by offering new options for sgRNA
design
http://chopchop.cbu.uib.no/ University of Bergen Labun etal.
(2016)
16 CRISPOR Web tool to nd guide RNAs from an
input sequence
http://crispor.tefor.net/ University of California (Santa
Cruz)
Haeussler etal.
(2016)
17 CCTop Online, intuitive user interface for
designing of guide RNAs
http://crispr.cos.uni-
heidelberg.de/index.html
University of Heidelberg Stemmer etal.
(2015)
18 sgRNA Scorer 2.0 Tool to identify sgRNA PAM sites for
gene sequence
http://crispr.med.harvard.
edu/sgRNA ScorerV2/
Wyss Institute for Biologically
Inspired Engineering at Harvard
Char etal.
(2017)
19 CRISPR-P 2.0 Web-services for computer-aided sgRNA
designing with minimal off-target
activity
http://crispr.hzau.edu.cn/
CRISPR2/
National Key Laboratory of Crop
Genetic Improvement and Center
for Bioinformatics, Huazhong
Agricultural University
Liu etal.
(2017a, b)
20 GuideScan Software for designing gRNA libraries
for various genomic regions
http://www.guidescan.com/ Leslie Lab and Ventura Lab Perez etal.
(2017)
21 CRISPR-GE Convenient, integrated toolkit to expedite
all experimental designs and analyses of
mutation for CRISPR/Cas/Cpf1-based
genome editing in plants and other
organisms
http://skl.scau.edu.cn Liu YG Lab, The Genetic
Engineering Laboratory of South
China Agricultural University
Xie etal. (2017)
S. Mehta et al.
435
S.No. Tool Specication Website URL address Provider References
22 CRISPR-Local High-throughput tool for designing
single-guide RNAs in plants and other
organisms
http://crispr.hzau.edu.cn/
CRISPR-Local/
National Key Laboratory of Crop
Genetic Improvement and Center
for Bioinformatics, Huazhong
Agricultural University
Sun etal. (2019)
23 CRISPR-PLANT
v2
Tool to predict off-target sites found in
unbiased genome-wide studies
http://www.genome.
arizona.edu/crispr2/
Arizona Genomics Institute Minkenberg
etal. (2019)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
436
23.4 CRISPR/Cas9 inRice forIncreasing Food Production
One way to address the global food demand is to increase the crop yield. However,
it affects various factors including selection of high-yielding/stress-tolerant culti-
vars, modication of existing cultivars, nutrient supply, water supply, and weed–
pest management. In the past 5years, the use of genome editing was in its infancy;
however, there are numerous successful reports currently in the literature.
23.4.1 Agronomic Traits Improvement
The most common way to improve the overall yield is to increase the grain number,
weight, and size (Sakamoto and Matsuoka 2008; Xing and Zhang 2010; Baltes etal.
2017). Genetically the underlying grain number, weight, and size are directly linked
with hundreds of genes and quantitative trait loci. Various major genes/QTLs have
been molecularly characterized and edited using the CRISPR/Cas9 system in rice.
Gene editing through CRISPR/Cas9in rice cultivar Zhonghua for loss of function
mutation in genes for grain number (Gn1a), grain size (GS3), panicle architecture
(DEP1), and plant architecture (IPA1). Mutated rice plants exhibit higher grain
Table 23.3 List of available CRISPR/Cas9 databases for plant systems
S.No. Database Purpose URL address Institution name References
1. CrisprGE A central
repository for
CRISPR/Cas-based
editing
http://crdd.
osdd.net/
servers/
crisprge/
CSIR-IMTECH,
India
Kaur etal.
(2015)
2. Cas-Database Genome-wide
gRNA library
design tool for
Cas9 nucleases
from Streptococcus
pyogenes
http://www.
rgenome.net/
cas-database/
Center for
Genome
Engineering,
Institute for Basic
Science, Korea
Park et al.
(2016)
3. Cpf1-
Database
Genome-wide
gRNA library
design tool for
Cpf1
http://www.
rgenome.net/
cpf1-
database/
Center for
Genome
Engineering,
Institute for Basic
Science, Korea
Park and
Bae (2017)
4. CRISPRlnc A manually curated
database of
validated sgRNAs
for lncRNAs
https://www.
crisprlnc.org/
Bioinformatics
Group of XTBG,
Chinese Academy
of Sciences
Chen etal.
(2019)
5. PGED (Plant
Genome
Editing
Database)
Database for
storing information
about CRISPR-
mediated mutants
in any plant species
http://
plantcrispr.
org/cgi-bin/
crispr/index.
cgi
Boyce Thompson
Institute
Zheng etal.
(2019)
S. Mehta et al.
437
number, larger grain size, and dense panicle, and ipa1 mutant shows lesser as well
as higher panicle number depending on mutation in the target site of miR156 (Li
etal. 2016a, b, c). Knockout mutation in Japonica rice, Kitaake cultivar for LAZY1
gene, exhibits higher tiller number (Miao etal. 2013).OsCAld5H1 gene knockout
by CRISPR/Cas9in rice leads to enrichment of G units in lignins and reveals its role
in the synthesis of non-c-p-coumaroylated S lignin units (Takeda et al. 2019).
Mutation in abscisic acid receptor family of genes, PYLs through CRISPR/Cas9in
rice, leads to improved growth and enhanced productivity (Miao etal. 2018). Grain
weight in rice is regulated by GW2, GW5, and TGW6. The multiple gene editing of
all three genes by CRISPR/Cas9in rice shows larger grain size as compared to non-
edited rice (Xu etal. 2016). Multiplex editing of genes Hd2, Hd4, and Hd5 medi-
ated by CRISPR/Cas9 leads to early maturity in rice (Li etal. 2017a, b). All these
results together provide information regarding already edited genes in various rice
cultivars for enhancing agronomic traits (Table23.5).
Table 23.4 List of commercial companies available for the implementation of CRISPR/Cas9
technology
S.No. Commercial companies Website link Headquarters
1 System Biosciences https://www.systembio.com/ California, United
States
2 Sigma–Aldrich https://www.sigmaaldrich.com/ Darmstadt, Germany
3 Integrated DNA
Technologies (IDT)
https://eu.idtdna.com/pages/ Iowa, United States
4 New England Bio Labs https://www.neb.uk.com/ Hertfordshire,
England
5 GeneCopoeia https://www.genecopoeia.com/ Maryland, United
States
6 DNA 2.0 https://www.atum.bio California, United
States
7 ORiGene https://www.origene.com/ Maryland, United
States
8 Eurons Genomics https://www.euronsgenomics.
co.in/
Karnataka, India
9 Genscript https://www.genscript.com/ New Jersey, United
States
10 Oxford Genetics https://www.scienceexchange.
com/
California, United
States
11 Cellectis https://www.cellectis.com/en/ New York, United
States
12 Pacic Biosciences https://www.pacb.com/ California, United
States
13 Addgene https://www.addgene.org/ Maryland, United
States
14 Macrogen http://www.macrogen.com/en/
main/index.php
Seoul, North Korea
15 ThermoFisherScientic https://www.thermosher.com/
in/en/home.html
Massachusetts,
United States
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
438
23.4.2 Enhanced Stress Tolerance/Resistance
A major bottleneck to the current rice productivity is due to the losses incurred by
pests, pathogens, and weeds. These biotic stresses are estimated to decline global
agricultural productivity by 40% (Mew etal. 1993; Oerke 2006; Savary etal. 2012).
In a favorable environment, blast disease causes 60–100% yield loss in rice-growing
area (Kihoro etal. 2013). Blast is one of the most devastating diseases in rice caused
by Magnaporthe oryzae (Zhang etal. 2014). Great efforts were made in the last few
decades for developing blast-resistant rice cultivar through the application of
genomics tools. Through conventional breeding approaches, blast-resistant rice has
been developed (Fukuoka etal. 2014; Ashkani etal. 2015). Conventional breeding
approaches are tedious in nature and need a longer duration. Other limitations like
the existence of pathogen variability and the emergence of new pathotype cause
breakdown of resistance barrier leading to severe disease infestation. Recent
advanced technologies like CRISPR/Cas9, TALEN, and ZFNs could be alternative
approaches for engineering rice genome for acquiring disease-resistant phenotype.
Blast disease-resistant phenotype is reported in rice by the disruption of ethylene-
responsive factor 922 (OsERF922) gene-mediated through CRISPR/Cas9 (Wang
etal. 2016). Targeted mutation through CRISPR/Cas9in the ethylene responsive
factor 922 provides blast disease resistance in rice (Liu etal. 2012). Expression of
OsSWEET13 gene in rice responsible for bacterial blight disease and indica rice,
IR24, with improved resistance for bacterial blight disease has been developed
through CRISPR/Cas9 knockout targeting promoter of OsSWEET13 (Zhou etal.
2015). By disrupting, promoter of OsSWEET14 gene by TALEN technology results
in resistance towards bacterial blight in rice (Li etal. 2012). TALEN technology is
0
100
200
300
400
500
600
700
800
900
2013 2014 2015 2016 2017 2018 2019
Number of Publicaons
Years
Articles (CRISPR-Cas9)
Articles (CRISPR-Cas9 in rice)
Fig. 23.1 The graph representing the number of publications per year related to CRISPR/Cas9
and CRISPR/Cas9 in rice by years 2013–2019. Keywords used in PubMed search included
CRISPR/Cas9 and rice. (Accessed on April 1, 2019)
S. Mehta et al.
439
Fig. 23.2 A timeline of key developments of CRISPR/Cas9 technology in rice
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
440
employed for modifying the promoter of Os09g29100 gene to nullifying EBEtal7
interaction, which could provide tolerance to BLB disease in rice (Cai etal. 2017).
TALENs targeting effector binding elements (EBEs) of AvrXa7 and Tal5 disrupt
their interaction with the susceptible gene Sweet14. The edited rice plants were
resistant to Xanthomonas infection. These technologies are quite helpful in develop-
ing rice cultivar with tolerant phenotype for Xoo (Li etal. 2012).
Fig. 23.3 Workow illustrating the successive steps for rice genome editing using CRISPR/Cas9
technology in rice
S. Mehta et al.
441
Table 23.5 Summary of CRISPR/Cas9-mediated genome editing in rice for agronomic traits
S.No. Gene Gene function Delivery method Cultivars
% mutations/
HR Mutant plant References
1LAZY1 Tiller number Agrobacterium-
mediated
Kitaake
(Japonica)
– More tiller number Miao etal.
(2013)
2Gn1a Grain number Agrobacterium-
mediated
Zhonghua
(Japonica)
42.5 Higher grain number Li etal. (2016a,
b, c)
3GS3 Grain size Agrobacterium-
mediated
Zhonghua
(Japonica)
57.5 Larger grain size Li etal. (2016a,
b, c)
4DEP1 Panicle architecture Agrobacterium-
mediated
Zhonghua
(Japonica)
67.5 Dense panicle Li etal. (2016a,
b, c)
5IPA1 Plant architecture Agrobacterium-
mediated
Zhonghua
(Japonica)
27.5 More panicle number Li etal. (2016a,
b, c)
6OsCAld5H1 Lignin synthesis Agrobacterium-
mediated
– 94 Enriched G-units in
lignin
Takeda etal.
(2019)
7PYLs Abscisic acid
receptor
Agrobacterium-
mediated
Nipponbare
(Japonica)
– Improved growth and
productivity
Miao etal.
(2018)
8GW2, GW5,
TGW6
Grain width and
grain size
Agrobacterium-
mediated
– – Larger grain size Xu etal. (2016)
9Hd2, Hd4, Hd5 Suppressor of
owering
Agrobacterium-
mediated
– – Early maturity Li etal. (2017a,
b)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
442
Rice production constrained by viral disease including rice tungro disease (RTD)
plays a major role in reducing rice production in rice-growing areas (Azzam and
Chancellor 2002; Muralidharan etal. 2003; Chancellor et al. 2006). Through the
development of near-isogenic lines (NILs), it is conrmed by the researchers that
resistance to RTSV and RTBV depend on the translation and in-frame mutation of
initiation factor 4 gamma (eIF4G) gene respectively (Lee etal. 2010; Macovei etal.
2018). In-frame mutation in eIF4G gene in rice confers resistant phenotype for
RTSV (Macovei et al. 2018).
In addition to pathogen resistance, weed management is also considered as a
critical factor in optimizing the crop yield. One of the effective ways is the appli-
cation of herbicides on the eld. Herbicide-resistant gene, bentazon-sensitive
lethal (BEL) knockout by CRISPR/Cas9, and biallelic mutated rice confer sen-
sitivity to bentazon. This trait could be successfully utilized for hybrid seed
production (Xu et al. 2014). CRISPR/Cas9-mediated gene replacement of
5-enolpyruvylshikimate- 3-phosphate synthase (EPSP) having the desired sub-
stitution gives glyphosate- resistant phenotype in rice (Li et al. 2016a, b, c).
Herbicide-tolerant rice cultivar is generated by mutation in the ALS gene by
genome editing (Li etal. 2016a, b, c; Sun etal. 2016). TALEN technology was
used for creating double-point mutation mediated through homology-directed
repair (HR) in OsALS rice gene (Li et al. 2016a, b, c). Rice ALS gene also
mutated at multiple points using CRISPR/Cas9 HR and edited rice plant shows
tolerance to bispyribac sodium (BS) spraying, and wild-type rice died after
36 days of herbicide spray (Sun et al. 2016). A point mutation generated in
acetolactate synthase (ALS) gene through CRISPR/Cas9 coupled with cytidine
deaminase confers tolerance to imazamox herbicide (Shimatani etal. 2017a, b)
(Table23.6).
Next to biotic stress, abiotic stresses are considered a factor that controls the rice
productivity (Mehta et al. 2019a, b). It includes ooding, drought, heavy metal
stress, metalloid stress, and heat stress(Dhakate etal. 2019). Rice plant is extremely
sensitive under low temperature especially during the early stage of development.
Therefore, the improvement of rice varieties for cold tolerance could signicantly
enhance productivity in rice. For enhancing cold tolerance in rice, TIFY1b and its
homology gene TIFY1a were edited through CRISPR/Cas9 (Huang et al. 2017).
Osmotic stress/ABA-activated protein kinase 2 (OsSAPK2) knockout mutant-
mediated by CRISPR/Cas9 exhibits higher sensitivity for drought and reactive oxy-
gen species than control rice plant (Lou etal. 2017).
23.4.3 Biofortification
Next to increasing food production, improving food nutritional value is the biggest
hurdle to the researchers. This demand has increased globally with the hike in
household incomes and food-related awareness in developing countries. As a result,
nowadays consumers require food with properties such as reduced cholesterol,
S. Mehta et al.
443
Table 23.6 Summary of CRISPR/Cas9-mediated genome editing related to various stresses
S.No. Gene Gene function Delivery method Cultivars
%
mutations/
HR Mutant plant References
1BEL Herbicide resistance Agrobacterium-
mediated
Rice cultivar
Nipponbare
2–16 Sensitive to bentazon Xu etal. (2014)
2SWEET13 Negative regulator of
blast resistance
Agrobacterium-
mediated
Indica rice IR24 – Resistance to bacterial
blight
Zhou etal.
(2015)
3ERF922 Negative regulator of
blast resistance
Agrobacterium-
mediated
Japonica rice
variety Kuiku131
42 Enhance blast resistance Wang etal.
(2016)
4EPSP Tolerance to glyphosate Biolistic
transformation
Rice variety
Nipponbare
2 Resistance to glyphosate Li etal. (2016a,
b, c)
5ALS Tolerance to bispyribac
sodium (BS)
Agrobacterium-
mediated
– – Tolerance to bispyribac
sodium (BS)
Sun etal. (2016)
6TIFY1a/TIFY1b Cold tolerance Agrobacterium-
mediated
Rice cultivar
Nipponbare
35–87.5 Tolerance to cold Huang etal.
(2017)
7ALS Resistance to herbicide
imazamox (IMZ)
Agrobacterium-
mediated
Rice cultivar
Nipponbare
3.41 Resistance to imazamox
(IMZ)
Shimatani etal.
(2017a, b)
8SAPK2 Tolerance to drought Agrobacterium-
mediated
Rice cultivar
Nipponbare
– Sensitive to drought Lou etal. (2017)
9eIF4G Susceptibility to rice
tungro virus
Agrobacterium-
mediated
Indica rice IR64 36.0–86.6 Resistance to rice tungro
spherical virus (RTSV)
Macovei etal.
(2018)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
444
Table 23.7 Successful reports of CRISPR/Cas9-mediated genome editing in rice biofortication
S.No. Gene Gene function Delivery method
Genotype
name
% of
mutation/HR Mutant plant Reference
1. SBEI, SBEIIb Starch debranching enzyme Agrobacterium-
mediated
Kitaake
(Japonica)
26.7–40 Enhanced amylose
content
Sun etal.
(2017)
2. Nramp5 Cadmium transporter Agrobacterium-
mediated
Indica 70–82.4 Low-grain cadmium
content
Tang etal.
(2017a, b)
3. OsPDS,
OsSBEIIb
Phytoene desaturase, starch
debranching enzyme
Agrobacterium-
mediated
– 20 Targeted mutations were
generated
Li etal.
(2017a, b)
4. BADH2 Betaine aldehyde
dehydrogenase
Agrobacterium-
mediated
– – Enhanced fragrance Shao etal.
(2017)
5. Waxy gene Starch synthesis Agrobacterium-
mediated
Japonica 82.76 Reduced amylose content Zhang etal.
(2018)
6. ISA1 Amylose synthesis Agrobacterium-
mediated
Zhonghua11 – Reduced amylose and
amylopectin content
Chao etal.
(2019)
S. Mehta et al.
445
biofortied whole grains, and low wax. As a result, various researchers have suc-
cessfully used CRISPR/Cas9 technology for biofortication especially in rice.
Loss of function mutation through CRISPR/Cas9 of waxy gene in rice has
reduced amylose content (Zhang etal. 2018). CRISPR/Cas9-mediated loss-of-func-
tion mutation of the starch debranching enzymes SBEI and SBEIIb has higher amy-
lose content and resistant starch (Sun etal. 2017). Knockout of ISA1 gene through
CRISPR/Cas9 in rice exhibit reduced amylose and amylopectin contents. The
mutant seeds were altered with shrunken endosperm and lesser grain weight (Chao
etal. 2019). Loss-of-function mutation of Nramp5 through CRISPR/Cas9 in rice
have low cadmium content when grown in cadmium-contaminated eld (Tang etal.
2017a, b). Targeted mutations through modied CRISPR/Cas9 (nCas9 containing
cytidine deaminase) for OsPDS and OsSBEIIb in rice were generated (Li et al.
2017a, b). Knockout of Badh2 gene mediated by CRISPR/Cas9 in rice exhibits
enhanced aroma (Shao etal. 2017). Table23.7 summarizes the successful reports of
rice biofortication.
23.5 Insights intothe CRISPR/Cpf1: AnAlternative
toCRISPR/Cas9
In addition to Cas9, scientists have reported other Cas family members for genome
editing in the last 5years. One of the promising members is Cpf1 (CRISPR from
Prevotella and Francisella1) (Zetsche etal. 2015). The mechanisms of CRISPR/
Cpf1 and CRISPR/Cas9 are compared in Table 23.8. In order to draw out more
information, the researchers are suggested to refer to the publications by Endo etal.
(2016), Wang etal. (2017), Xu etal. (2017), and Jun etal. (2019).
All the successful reports regarding the application of CRISPR/Cpf1in rice are
enlisted in Table23.9.
Table 23.8 Comparison of Cas9- and Cpf1-mediated editing
Attributes Cas9 Cpf1 (Cas12a)
gRNA components tracrRNA and crRNA crRNA
gRNA length (bp) ≤100 ≤43
Type of ends produced Blunt ends Sticky ends
Type of overhang
generated
No 5′ overhang
Target PAM site G-rich T-rich
PAM sequence 5′-NGG-3′5′-TTTN-3′
Cutting site 3–4bp upstream to the PAM
site
18–24bp downstream to the PAM
site
RNase III required Yes No
Off-target effects Yes, comparatively higher Yes, comparatively low
Nickase generation Possible, already done Impossible
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
446
Table 23.9 Summary of CRISPR/Cpf1-employed editing in rice
S.No. Tool Gene Gene function Cultivars Delivery method
%mutations/
HR (%) Mutant plant Reference
1 CRISPR/
FnCpf1
DL (drooping
leaf)
Midrib formation Japonica rice
cultivar
Nipponbare
Agrobacterium-
mediated
8.3–60 All mutants show a
loss of midrib leading
to drooping leaf
phenotype
Endo etal.
(2016)
2 CRISPR/
FnCpf1
ALS
(Acetolactate
synthase)
Involved in the
synthesis of
branched chain
amino acids
Japonica rice
cultivar
Nipponbare
Agrobacterium-
mediated
15–60 Loss of ALS activity
leading to lethality
Endo etal.
(2016)
3 CRISPR/
FnCpf1
RLK-798,
RLK-799,
RLK-802,
RLK-803
Receptor like
kinases
Japonica rice – 43.8–75 – Wang etal.
(2017)
4 CRISPR/
FnCpf1
NAL1 Phosphate (Pi)
accumulation
Japonica Rice Agrobacterium-
mediated
– Enhanced phosphate
accumulation
Hu etal.
(2017)
5 CRISPR/
FnCpf1
LG1 Legule formation Japonica Rice Agrobacterium-
mediated
– No ligule formation Hu etal.
(2017)
6 CRISPR/
LbCpf1
BEL-230,
BEL-240,
BEL-250,
BEL-260
Bentazon-sensitive
lethal
Japonica rice – 40–60 – Wang etal.
(2017)
7 CRISPR/
FnCpf1 and
CRISPR/
LbCpf1
MPK2, MPK5 Mitogen-activated
protein kinase
Japonica rice
Zhonghua 11
Agrobacterium-
mediated
9–32 – Ding etal.
(2018)
S. Mehta et al.
447
8 CRISPR/
FnCpf1 and
CRISPR/
LbCpf1
PDS Phytoene
desaturase
Japonica rice
Zhonghua 11
Agrobacterium-
mediated
– Albino Ding etal.
(2018)
9 CRISPR/
FnCpf1 and
CRISPR/
LbCpf1
DEP1 Dense and erect
panicle
Rice
protoplast
PEG-CaCl2-
mediated
90 Scattered panicle Zhong
etal.
(2018)
10 CRISPR/
FnCpf1 and
CRISPR/
LbCpf1
ROC5 Leaf rolling Rice
protoplast
PEG-mediated – Outcurve rolled
leaves
Zhong
etal.
(2018)
11 CRISPR/
LbCpf1
EPFL9 Regulation of
stomatal density
and patterning
Indica rice
cultivar IR64
Agrobacterium-
mediated
– Altered stomatal
pattern and density
Yin etal.
(2019)
23 CRISPR/Cas9-Edited Rice: ANew Frontier forSustainable Agriculture
448
23.6 Conclusion
In the past ve decades, crop improvement via traditional breeding has signicantly
contributed to acquiring food security for the every second whopping human popu-
lation. However, various developments require more manpower, time duration,
efforts along with high chance of failures in getting the “desirable traits”.
Additionally, other conventional technologies like chemical mutagenesis, soma-
clonal variation, in vitro tissue culture and physical irradiation have also multiple
loopholes. For increasing crop production under the changing climate as well as
fullling the caloric and nutritional demands of mankind, the most recent,
advanced nuclease-based technologies have emerged as the most suitable candidate
in many crops including rice. Among all these technologies, the CRISPR/Cas9 tool
is more precise, easy to handle, and also employed for avoiding backcrossing of a
huge number of inbred lines. The varietal development using CRISPR/Cas9 tech-
nology consumes less time and is easy to introduce/restore desired changes in the
existing elite rice germ plasm. Recently, multiple genes have been stacked together
to get the desired phenotype in rice. Additionally, due to the advances like base edit-
ing, gene targeting, and DNA-free genome editing, the rice researchers have afr-
matively taken a big leap towards the biggest milestone, i.e., super rice generation.
Furthermore, due to the technical advances in the post- genomic era, the researchers
have characterized a plethora of negative regulatory genes, SNPs, and QTLs for
various traits. Taking these points, we hope that our children will be eating the
socially accepted, highly nutritious super rice in the long run in the future.
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