Received: 18 April 2017 | Accepted: 20 April 2017

DOI: 10.1002/jcp.25970

MINI-REVIEW

CRISPR/Cas9: An RNA-guided highly precise synthetic tool

for plant genome editing

Yeliz Demirci1 | Baohong Zhang2 | Turgay Unver1,*

1 Izmir International Biomedicine and Genome
Institute (iBG-izmir), Dokuz Eylul University,
Izmir, Turkey
2 Department of Biology, East Carolina
University, Greenville, North Carolina
Correspondence
Turgay Unver, PhD, International Biomedicine
and Genome Institute (iBG-izmir), Dokuz Eylül
University, Balcova 35340 Izmir, Turkey.
Email: turgay.unver@deu.edu.tr
Funding information
Turkish Academy of Science (TUBA) with grant
type GEBIP and TUBITAK, Grant number:
113O016; Cotton Incorporated, Grant number:
15-770
CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally
used by bacteria for immune defence. In the past years, it has been quickly employed and
modified to precisely edit genome sequences in both plants and animals. Compared with the well-
developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases
(TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher
targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for
knockout of an individual gene as well as insertion of one gene and/or control of gene
transcription. Studies have shown that CRISPR/Cas9 is a great tool to edit many genes in a variety
of plant species, including the model plant species as well as agriculturally important crops, such
as cotton, maize, wheat, and rice. CRISPR/Cas9-based genome editing can be used for plant
functional studies and plant improvement to yield, quality, and tolerance to environmental stress.
KEYWORDS
CRISPR/Cas9, crop improvement, genome editing, plant breeding

1 | ORIGIN OF THE CRISPR/CAS9 SYSTEM
Increases in human population and climate change lead to certain
significant problems on meeting food need and sustainability of
agricultural production. In order to overcome these challenges, various
classical plant breeding techniques have been employed to increase the
amount and quality of yield. However, classical plant breeding techniques
are not enough to precisely manipulate target genes encoding
corresponding traits. Over the last decades, improved plant breeding
methods, including biotechnology and molecular genetics approaches,
have been employed in the plant breeding programs to facilitate plant
genome manipulation and desired agronomic trait selection. Additionally,
emerging genome editing technologies such as Zinc Finger Nucleases
(ZFNs) (Zhang et al., 2010), transcription activator-like effector nucleases
(TALENs) (Li, Liu, Spalding, Weeks, & Yang, 2012) are being used to edit
plant genomes. Recently, the Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)-Cas (CRISPR-associated protein) system
has been discovered and adapted for targeting genome editing to
manipulate desired traits in various organisms, including plants (Alagoz,
Gurkok, Zhang, & Unver, 2016; Gratz et al., 2014; Hwang, Fu, Reyon,
Maeder, & Kaini, et al., 2013).
*Current address: Turgay Unver, Egitim Mah. Ekrem Guer Sok. No:26/3,
35340 Balcova, Izmir, Turkey.
In 1987, CRISPR was discovered in Escherichia coli genome by
Ishino, Shinagawa, Makino, Amemura, and Nakata (1987) while
identifying the iap gene product. They incidentally cloned a part of
CRISPR region while cloning iap gene; their results showed that the
arrangement of repeats is consecutive throughout the bacterial
genome. Following that study, CRISPR sequences were also found
in other organisms, such as Haloferax mediteranii, an archea
(Mojica, Ferrer, Juez, & Rodríguez-Valera, 1995). In 2000, a large
number of regularly spaced repeats with a related function were
identified (Mojica, Díez-Villaseñor, Soria, & Juez, 2000). These
repetitive DNA sequences were only existed in prokaryotes but
not in viruses and eukaryotes, which were named as CRISPR
(Jansen, Embden, Gaastra, & Schouls, 2002). At that time, four
CRISPR-associated (Cas) genes (Cas1-4) were identified in CRISPR-
positive prokaryotes (Jansen et al., 2002). Following this report,
different Cas protein families and multiple CRISPR/Cas subtypes
were reported (Haft, Selengut, Mongodin, & Nelson, 2005). In
2005, three independent research groups demonstrated that some
CRISPR spacers are originated from phage and plasmid DNA
(Bolotin, Quinquis, Sorokin, & Ehrlich, 2005; Mojica, Díez-
Villaseñor, García-Martínez, & Soria, 2005; Pourcel, Salvignol, &
Vergnaud, 2005). However, until 2007, its function was still
unclear. Barrangou and his colleagues showed firstly an experi-
mental evidence that CRISPR provides acquire resistance against a

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2 |
virus in Streptococcus thermophilus by adding a genomic part of a
virus into its CRISPR locus (Barrangou et al., 2007a). The immune
defence of CRISPR interference limits horizontal gene transfer in
staphylococci by targeting DNA (Marraffini & Sontheimer, 2008);
CRISPR RNAs (crRNAs) play the guidance role in CRISPR
intervention (Brouns et al., 2008). As a part of bacterial immune
system, Cas proteins cleave bacteriophage and plasmid DNA at
precise sites (Garneau et al., 2010). In 2011, trans-activating
crRNAs (tracrRNAs) were identified (Deltcheva et al., 2011) and
CRISPR/Cas system was firstly transferred between different
organisms, such as from E. coli to S. thermophilus (Sapranauskas
et al., 2011). Conclusively, CRISPR/Cas system become a targeted
genome-editing tool by demonstrating the functions of tracrRNA,
crRNA, and Cas9 protein which generate a double-strand break
(DSB) in the DNA sequence including homology to the crRNA
spacer region of the S. pyogenes CRISPR/Cas9 system in 2011
(Jinek et al., 2012). Later, CRISPR/Cas9 mediated site-specific
DNA mutagenesis were first reported in mammalian genomes
including human (Cong et al., 2013; Mali et al., 2013). Additionally,
CRISPR/Cas9-mediated genome editing was successfully demon-
strated in zebrafish (Hwang, Fu, Reyon, Maeder, & Tsai, et al.,
2013) and bacterial cells (Jiang, Bikard, Cox, Zhang, & Marraffini,
2013). At the same time, CRISPR/Cas9 technology were employed
for plant genome editing using different transformation methods
such as agroinfiltration and protoplast transfection with different
repair mechanisms (NHEJ and HDR to create insertions and
deletions) in model plant species Arabidopsis thaliana and Nicotiana
benthamiana, and crop plant species Oryza sativa (Feng et al.,
2013; Gao et al., 2013; Li et al., 2013; Nekrasov, Staskawicz,
Weigel, Jones, & Kamoun, 2013; Xie & Yang, 2013b). Currently, a
variety of plant species were modified by CRISPR/Cas9 technol-
ogy, which include Brassica oleracea (Lawrenson et al., 2015), Citrus
sinensis (Jia & Wang, 2014a), Cucumis sativus (Chandrasekaran
et al., 2016), Glycine max (Jacobs, LaFayette, Schmitz, & Parrott,
2015), Gossypium hirsutum (Li, Unver, & Zhang, 2017), Hordeum
vulgare (Lawrenson et al., 2015), Lactuva sativa (Woo et al., 2015),
Lotus japonicus (Wang, Wang, & Tan, et al., 2016), Marchantia
polymorpha (Sugano et al., 2014), Medicago truncatula (Michno
et al., 2015), Nicotiana attenuate (Woo et al., 2015), N. tabacum
(Baltes, Gil-Humanes, Cermak, Atkins, & Voytas, 2014), Papaver
somniferum (Alagoz et al., 2016), Petunia hybrida (Subburaj et al.,
2016), Physcomitrella patens (Collonnier et al., 2016), Populus
tomentosa (Fan et al., 2015), P. tremula (Zhou, Jacobs, Xue, Harding,
& Tsai, 2015), Solanum lycopersicum (Ron et al., 2014), S. tuberosum
(Wang, & Zhang, et al., 2015), Sorghum bicolor (Jiang, Zhou, et al.,
2013), Taraxacum kok-saghyz (Iaffaldano, Zhang, & Cornish, 2016),
Triticum aestivum (Gao et al., 2013) Vitis vinifera (Ren et al., 2016),
and Zea mays (Liang, Zhang, Chen, & Gao, 2014).
1.1 | Structural properties of CRISPR/Cas9 system
CRISPR/Cas systems play a critical role in the prokaryotic adaptive
immune system in archaea and bacteria against phage and virus attacks
(Bhaya, Davison, & Barrangou, 2011; Terns & Terns, 2011). Each native
DEMIRCI ET AL.
CRISPR/Cas system composes of a cluster of CRISPR-associated (Cas)
genes encoding RNA-guided endonucleases, identical palindromic
repeats, and unique noncoding short RNAs, called as “spacer” created
with the insertion of invader nucleic acids (short variable sequences,
named as, protospacers). Once the cell attacked, the new spacers
derived from protospacers are located between the identical palin-
dromic repeats. The spacers are used as recognition components that
make it possible to the invaded cell to remember and to destroy foreign
DNA fragments. Although the CRISPR/Cas system exists in both
archaeal and bacterial genomes, archaea has more CRISPR/Cas systems
in their genomes (87% of their genomes) than bacteria (50% of genomes)
(Makarova, & Haft, et al., 2011; Westra, Buckling, & Fineran, 2014).
CRISPR-based defence mechanism consists of three phases:
adaptation, expression, and interference. Adaptation phase is the first
one and it involves the acquisition of new spacers in the CRISPR-array
in chronological arrangements. In the expression phase, the Cas genes
and precursor CRISPR-RNA (pre-crRNA) are expressed. Cas proteins
and other factors (in type II RNase III) are employed to generate mature
cr-RNA from pre-crRNA. By integrative effects of cr-RNA and Cas
proteins, the targeted region is remembered and cleaved in the last
interference phase (Bortesi & Fischer, 2015). The protospacer
adjacent motif (PAM), also called CRISPR motif, adjacently located
in the target sequence is associated with each protospacer. PAM is a
conserved sequence of the invader virus or phage but is not a
component of the bacterial CRISPR locus (Barrangou et al., 2007b;
Brouns et al., 2008; Marraffini & Sontheimer, 2008). Target
recognition of the Cas9 needs a conserved dinucleotide-containing
PAM sequence upstream of the crRNA-binding site (Jinek et al., 2012).
While recognition of PAM, Cas9 can not successfully bind to target
DNA for effective cleavage. PAM is also a unique sequence that
discriminates bacterial self or non-self DNA (Mojica, Diez-Villasenor,
Garcia-Martinez, & Almendros, 2009) and helps bacteria to protect self
DNA from cleavage of nucleases. In various type of CRISPR systems,
Cas orthologs require different PAM sequences. For instance, S.
pyogenes Cas9 targets DNA containing PAM 5′-NGG (Jinek et al.,
2012); however, S. thermophilus Cas9 requires the PAM sequence of
5′-NNAGAA (Cong et al., 2013; Garneau et al., 2010) or 5′-NGGNG
(Gasiunas, Barrangou, Horvath, & Siksnys, 2012) and Neissseria
meningiditis Cas9 requires 5′-NNNNGATT (Zhang et al., 2013). After
its initial discovery, two classes (class 1–2) and three main types (type
I–III) of CRISPR/Cas systems were identified in host organisms
(Makarova, Aravind, Wolf, & Koonin, 2011; Makarova, Haft, et al.,
2011). Currently, two additional putative new types and five new
subtypes were discovered by a comparative analysis of available
genomic data (Makarova et al., 2015). In order to cleave foreign nucleic
acid strand, while class 1 system utilizes multiple Cas proteins, class 2
system uses a single large Cas protein. Class 1 is separated as I, III, and
IV; class 2 is divided into types II and V. Three main types of CRISPR
systems can be recognized in the presence of unique proteins: Cas3 for
type I and Cas9 and Cas10 for type II and type III, respectively
(Makarova, Haft, et al., 2011). In most known CRISPR/Cas systems,
Cas1 and Cas2 proteins work in adaptation phase to make it possible
insert spacers into CRISPR arrays. Type I system uses Cas5 or Cas6 to
process the pre-crRNA. Type III system also utilizes Cas6 for the same
DEMIRCI ET AL.
F I G UR E 1 Schematic representation of CRISPR/Cas9-mediated
genome editing. (a) tracrRNA (green) and crRNA (magenta) with
protospacer element are fused via a linker loop (orange) to form a
sgRNA. (b) To function CRISPR/Cas9 system, sgRNA, and Cas9
endonuclease generate a complex. (c) Cas9:sgRNA complex can
recognize the target site in the presence of PAM sequence
upstream of the site. The catalytic activity of Cas9 results in
double-stranded breaks (DSBs) activating host-mediated DNA repair
pathways. (d) DSBs can be fixed via NHEJ and HDR mechanisms
leading to knock-out or knock-in a gene at desired sites on genomic
DNA
purpose but 3′ end stimulation is performed by an unclear factor.
RNase III and tracrRNA are used in type II systems for maturation of
crRNA (Figure1a) (Rath, Amlinger, Rath, & Lundgren, 2015). Type II,
which is derived from S. pyogenes immune system is one of the best-
characterized system underlying the current editing technology and
commonly named as CRISPR. As a modification tool, CRISPR/Cas9
system includes two main components: an endonuclease Cas9 to
create double-stranded DNA (dsDNA) breaks and a chimeric non-
coding RNA (Figure 1b) (Jinek et al., 2012). This chimera consisting of
CRISPR RNA (crRNA): trans-activating crRNA (tracrRNA) is also called
guide RNA (gRNA or single guide RNA, sgRNA, sometimes synthetic
guide RNA) to direct Cas9. Target recognition of Cas9 is performed in
the presence of a “seed” sequence in the repeat/spacer-derived short
crRNA and a PAM sequence for the commonly used SpCas9 5′NGG′3
adjacent to the binding region in the target DNA (Jinek et al., 2012).
The tracrRNA is complementary to the repeat regions of crRNA
precursor transcripts and it triggers crRNA maturation from pre-
crRNA by the conserved endogenous RNase III and induces
| 3
crRNA-guided DNA cleavage by Cas9 (Deltcheva et al., 2011). As
reported by Nishimasu et al. (2014), the crystal structure of S. pyogenes
Cas9 (SpCas9), an RNA-guided DNA endonuclease has two-lobed
structure consisting of a target recognition (REC) lobe that recognizes
and binds to sgRNA:DNA heteroduplex and a nuclease (NUC) lobe for
cleavage of the target strand. While three regions of REC lobe includes
bridge helix, REC1, and REC2 domains, the NUC lobe consists of RuvC
(RuvC I–III motifs), HNH and PI (PAM-interacting) domains (Figure 1c).
The repair mechanism of genome determines the type of the
manipulation. DSBs generated by Cas endonuclease, can be repaired
via non-homologous end joining (NHEJ) and homologous recombina-
tion (HR, also called homology-directed repair, HDR) (Figure 1d). In an
error-prone repair mechanism, NHEJ, the broken ends are brought
together and rejoined by DNA ligation without the requirement of
homologous template for repair of the DNA breaks. Repairs usually
result in the loss of nucleotides at the target site. If necessary, a new
donor DNA fragment can be inserted by generating two separate
DBSs at one loci. If DSBs occur in different chromosome, it can be
afforded by translocation (Samanta, Dey, & Gayen, 2016). The
presence of a pair sgRNA, NHEJ mechanism might result with large
deletions. For example, using co-expressed two sgRNAs, effective
large chromosomal deletions achieved by NHEJ repair pathway in rice
(Xie, Zhang, & Yang, 2014). In a simple way, homologous recombina-
tion-based repair mechanism HDR occurs when a template that is
homologous with DSB site is available. Since any type of targeted
mutation such as targeted knock-in (Schiml, Fauser, & Puchta, 2014),
gene replacement (Li et al., 2013), and DNA correction is able to
achieved in plants via HDR, this type of repair is more remarkable for
plant engineering. For designing sgRNAs, several online tools are
available for different organisms, which include CHOPCHOP (https://
chopchop.rc.fas.harvard.edu) and CRISPR Design (https://crispr.mit.
edu). Some of them offer available plant databases enabling to a design
of sgRNAs and to explore possible off-target sites (Stemmer,
Thumberger, del Sol Keyer, Wittbrodt, & Mateo, 2015) (Table 1).
For example, CRISPR-PLANT (https://www.genome.arizona.edu/
crispr/) was established to help researchers for designing plant
genome editing CRISPR/Cas9 systems (Xie, Zhang, & Yang, 2014).
Recently, an online web tool (https://stuparcrispr.cfans.umn.edu/
CRISPR/) was developed for quick identification of CRISPR/Cas9
target loci in soybean. Utilizing the web tool, soybean codon-
optimized CRISPR/Cas9 platform was designed to create DSBs into
the target loci in G. max and M. truncatula, which further facilitated
targeted mutagenesis in legume and other plant species (Michno et al.,
2015). Cas9 and sgRNA can be introduced into the target cells by
various techniques, including Agrobacterium-mediated transformation,
and biolistic transformation (Miao et al., 2013). The efficiency of
CRISPR/Cas9-medaited genome editing is depended on the usage of
promoters for sgRNA and the stability of Cas9 enzyme expression. In
plants, RNA Polymerase II-dependent promoters such as CaMV35S
have been used for efficient Cas9 expression, RNA pol III dependent
promoters such as U3 or U6 promoters were benefited for sgRNA
expression (Kumar & Jain, 2015). Expression levels of sgRNAs driven
by endogenous promoters are higher than exogenous ones (Sun et al.,
2015). Additionally, U6 promoters derived from dicotyledonous or
4 | DEMIRCI ET AL.

TABLE 1 Summary of available web tools for designing of CRISPR/Cas system

Availability of plant
Name of the software/Website link databases Ref. Year
https://www.rgenome.net/cas-designer/ Yes Park, Kim, and Bae (2016) 2016
CRISPR-DO https://cistrome.org/crispr/ No Ma et al. (2016) 2016
CRISPy https://crispy.secondarymetabolites.org/#/input No Blin, Pedersen, Weber, and Lee (2016) 2016
phytoCRISP-Ex https://www.phytocrispex.biologie.ens.fr/CRISP-Ex/ No Rastogi, Murik, Bowler, and Tirichine 2016
(2016)
CCTop—CRISPR/Cas9 target online predictor https://crispr.cos.uni- Yes Stemmer et al. (2015) 2015
heidelberg.de/
https://stuparcrispr.cfans.umn.edu/CRISPR/ Yes Michno et al. (2015) 2015
https://research.microsoft.com/en-us/projects/azimuth/ No Fusi, Smith, Doench, and Listgarten 2015
(2015)
CRISPRseek https://www.bioconductor.org/packages/release/bioc/ No Zhu, Holmes, Aronin, and Brodsky 2014
html/CRISPRseek.html (2014)
CRISPR-PLANT https://www.genome.arizona.edu/crispr/ Yes Xie, Zhang, et al. (2014) 2014
SgRNAcas9 https://www.biootools.com/col.jsp?id = 103/ No Xie, Shen, et al. (2014) 2014
CasOT https://eendb.zfgenetics.org/casot Yes Xiao et al. (2014) 2014
https://gt-scan.braembl.org.au Yes O’Brien and Bailey (2014) 2014
CHOPCHOP https://chopchop.cbu.uib.no/ Yes Montague, Cruz, Gagnon, Church, and 2014
Valen (2014)
CRISPR-P https://cbi.hzau.edu.cn/crispr Yes Lei, Liu Li, Xing, and Chen (2014) 2014
E-CRISP https://www.e-crisp.org/E-CRISP/designcrispr.html Yes Heigwer, Kerr, and Boutros (2014) 2014
https://www.broadinstitute.org/rnai/public/analysis-tools/sgrna- No Doench et al. (2014) 2014
design
Cas-OFFinder https://www.rgenome.net/cas-offinder/ Yes Bae, Park, and Kim (2014) 2014
CRISPRdirect https://crispr.dbcls.jp/ Yes Naito, Hino, Bono, and Ui-Tei (2014) 2014
CRISPR Design Tool https://crispr.mit.edu/, https://www.genome- Yes Hsu et al. (2013); Ran et al. (2013) 2013
engineering.org

monocotyledonous species can be used for sgRNA expression in only
corresponding plant clades being dicot or monocot plants (Mao,
Botella, & Zhu, 2016). In eukaryotic organisms including plants, nuclear
localization signals (NLS) must be combined into the Cas9 for
efficiently delivering the CRISPR/Cas system into the nuclei (Belhaj,
Chaparro-Garcia, Kamoun, & Nekrasov, 2013).
1.2 | Difference between the classical breeding and
new plant breeding techniques (NPBT)
Plant breeders have been attempting to manipulate the traits that
affect plant yield, quality, and tolerance to environmental abiotic and
biotic stresses. Traditional plant breeding techniques are majorly
based on homologous recombination between chromosomes. Selec-
tion and planned interbreeding (crossing) have been used to improve
food and industrial crops for many centuries. However, due to the
fact of many limitations, including polyploidy, heterozygosity, self-
incompatibility, long generation periods, and time consuming, the new
plant breeding techniques (NPBTs) has become necessary. Since
ancient times, plant breeders have been trying to identify and select
desired traits and combine these characters into one individual plant
using some conventional techniques. The traditional breeding requires
continuous evaluation and selection for several generations (Chahal &
Gosal, 2002). Plant breeding has been used as a tool in order to
minimize the adverse effects of the excessive amount of cadmium (Cd)
intake in humans. While the selection is an effective method in
reducing the Cd concentration, generating of a new cultivar is always
time-consuming. For example, in Canada, developing a durum wheat
cultivar with low-Cd concentration was achieved in 10 years
(Schubert, Neubert, Schierholt, Sümer, & Zörb, 2009). Hybridization
is another conventional and the most commonly used breeding
technique, which combines the traits from different cultivars by
crossing. According to how many plant crossing, it is classified single
cross (two plants) or multiple crosses (three or more plants). As an
example of interspecific hybridization method, ADT-37, a rice variety
with resistance to many pests, and diseases, was bred via the crossing
of O. japonica and O. indices which took for 7 years (Soundararaj,
Sivasubramanian, & Chelliah, 1987). Although traditional methods
have bred many new varieties, due to their limitations such as time-
consuming, needing a large area for growing, and more expensive, a
new approach relying on the usage of engineered nucleases has been
quickly developed in the past decade. These powerful methods,
including zinc-finger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs), are derived from zinc-finger (ZF), and
transcription activator-like effector (TALE) proteins. Both of them are
chimeric proteins composing of an engineered sequence-specific DNA
DEMIRCI ET AL.
binding domain and a non-specific DNA cleavage module from FokI, a
type II restriction endonuclease (Kim, Cha, & Chandrasegaran, 1996).
This sequence-specific DNA binding domain can be engineered to
recognize a specific region in both methods, DNA cleavage module is
not engineered in both ZFNs and TALENs. They can successfully target
genome modifications; however, both of them have some limitations
in designing of the constructs. ZFNs is the first enzyme for genome-
editing plant genomes in Arabidopsis (Zhang et al., 2010), rice (Shukla
et al., 2009), tobacco (Townsend et al., 2009), and maize (Shukla et al.,
2009). Zinc-fingers (ZFs) are members of the transcription factor
family and each ZF can recognize three to four bases of DNA
sequence. A ZFN is a heterodimer and its each subunit includes a zinc
finger domain composing of three to six ZFs located in N-terminus
enabling to recognize a total of 18–36 nucleotides and a
FokI endonuclease domain introducing a DSB in the targeted sequence
that triggers DNA repair mechanisms (Kim & Kim, 2014).
FokI restriction endonuclease is sit in the C-terminus (Li, Wu, &
Chandrasegaran, 1992). Currently, various gene disruption, correction,
and edition applications were achieved in plants through ZFNs. ADH1
and TT4 genes were disrupted in A. thaliana by using ZFNs; the genetic
mutagenesis frequency was about 7% or 16%, respectively (Zhang
et al., 2010). ZFN technology was also used for editing IPK1 gene in
maize (Shukla et al., 2009) and for correcting SuRA and SurRB genes in
tobacco (Townsend et al., 2009).
TALEs are naturally occurring proteins derived from plant
pathogenic bacterial genus Xanthomonas (Gaj, Gersbach, & Barbas,
2013). As ZFNs, TALEs also have FokI activities. TALENs have two
domains: DNA-binding domain located in N-terminus and DNA-
cleavage domain. In the DNA-binding domain, there is a highly
conserved repeated 33–34 amino acid sequence. Polymorphisms
at 12 and 13 positions, also called Repeat Variable Diresidue
(RVD), allow specific sequence recognition of TAL effectors
(Moscou & Bogdanove, 2009). Bacterial blight susceptibility
gene, Os11N3 (also named as OsSWEET14), was disrupted in O.
sativa by TALEN-mediated genome editing (Li et al., 2012).
GmPDS11 and GmPDS18 genes were manipulated in G. max by
TALEN with a target efficiency range of 17.5–21.1% (Du et al.,
2016). All of the ZFNs, TALENs, and Cas9 nuclease technologies
are able to manipulate genome sequences in a specific region by
generating DSBs and triggering genome editing via DNA repair
mechanisms including both NHEJ and HDR. However, there are
some differences between these three approaches. First of all, in
order to target the desired sequence, while ZFN and TALEN
technologies use protein-DNA interactions (Li et al., 2012; Zhang
et al., 2010), Cas9 system uses simple DNA-RNA base pairing
interaction between the target DNA and a guide RNA (Jinek et al.,
2012). In ZFN-mediated genome editing, each ZF module can bind
to a 3-nt sequence, but each subunit of TALENs is associated with
a single base. Additionally, CRISPR/Cas9 system has no methyl-
ation sensitivity, however, ZFNs and TALENs are sensitive to
methylated DNA (Mao, Botella, et al., 2016). Compared with ZFNs
and TALENs, CRISPR/Cas9 has more advantages in genome editing
including the multiplex genome editing, simpler to design and
implement, higher targeting efficiency, and less expensive.
| 5
2 | A PP L I C A T I O N OF C R I S P R / C A S I N
MODEL AND CROP PLANT S PECIES
Shortly after the first demonstration of CRISPR/Cas defence system as
a genome editing technology, various research groups reported the
applications of CRISPR/Cas9 in different plant species (Table 2). While
the initial plant studies of CRISPR/Cas technology were mostly about
the optimization of the system to plant genomes, the following ones
were mostly related with development and customization of the
technology to increase its efficiency for different plant species or
different traits. CRISPR/Cas was first used to edit plant genome
sequences in A. thaliana, N. benthamiana, and O. sativa. Different
transformation methods have been employed for transforming
CRISPR/Cas system into plant cells, which include Agrobacterium-
mediated transformation and protoplast transfection. CRISPR/Cas-
mediated genome editing can be used to generate specific mutations
for both dicot and monocot plants (Feng et al., 2013; Gao et al., 2013;
Li et al., 2013; Nekrasov et al., 2013; Xie & Yang, 2013b). At the
beginning, CRISPR/Cas-mediated genome editing were mostly used to
target one or two gene loci at the same time. However, in order to
target more than one loci simultaneously, designed multiplex CRISPR/
Cas9 systems can offer the co-expression of multiple sgRNAs. A binary
vector containing gRNAs for six genes targeting PYR1, PYL1, PYL2,
PYL4, PYL5, and PYL8 with three different RNA polymerase III-
dependent promoters (AtU3, AtU6, and At7SL-2) were constructed
for multiple gene knockout in Arabidopsis. The results show that this
system can knockout of all tested genes with mutagenesis frequency
of 13–93% in T1 transgenic lines (Zhang et al., 2015). The rice genome
was targeted by two sgRNAs for OsYSA and one sgRNA for OsROC5
gene under OsU6 and OsU3 promoters in the same construct,
∼200 bp deletion were detected (Qi et al., 2015).
CRISPR/Cas9-mediated genome editing has produced a lot of
observed phenotype. GC contents of designed sgRNAs affected the
efficiency of gene knockout (Pan et al., 2016). sgRNAs with more than
50% GC content resulted in high efficiency (84–100%), and a relatively
low GC content (40%) has a lower efficiency (73%). In tomato (S.
lycopersicum L.), sgRNAs were designed to target phytoene desaturase
(SlPDS) and phytochrome interacting factor (SlPIF4) genes. Knockout of
the SlPDS gene resulted in photobleaching and clear albino phenotypes.
PDS and PDR6 genes of tobacco (N. tabacum) were successfully edited
by CRISPR/Cas9 via protoplast transfection; the mutation frequencies
were up to 20.3% and 87.5%, respectively. The phenotypic changes
were measured in the etiolated leaves for the psd mutant and more
branches for the pdr6 mutant (Gao, Wang, et al., 2015).
CRISPR/Cas system is not only employed to knock-out an
individual gene but also to knock-in an gene even for gene
replacement. Gene replacement by using CRISPR/Cas system enables
the insertion, elimination, and replacement of a specific gene
(Schaeffer & Nakata, 2015). CRISPR/Cas9-mediated gene replace-
ment was achieved in the rice endogenous gene 5-enolpyruvylshiki-
mate-3-phosphate synthase (OsEPSPS); the efficient of heritable gene
replacement has been observed at a frequency of 2.0% and the
generated rice plants carrying OsEPSPS gene have glyphosate-
resistant (Li, Meng, et al., 2016). CRISPR/Cas9 genome editing is
6 | DEMIRCI ET AL.

TABLE 2 Applications of CRISPR/Cas9-mediated genome editing in plants

gRNA
Species Delivery method promoter Target gene(s) Ref. Year
Arabidopsis Agrobacterium-mediated na ADH1 Schiml, Fauser, and Puchta 2016
thaliana transformation (2016)
Arabidopsis Agro-transformation by floral dip At(MGE1p ETC2, CPC, and TRY Eid, Ali, and Mahfouz 2016
thaliana MGE2p, (2016)
MGE3p)
Arabidopsis Agro-transformation by floral dip AtU6 ABP1 Gao, Chen, Dai, Zhang, and 2016
thaliana Zhao (2016)
Arabidopsis Agro-transformation by floral dip AtU6 AtSH3P3 Kim et al. (2016) 2016
thaliana
Arabidopsis Agro-transformation by floral dip AtU6 AP1, TT4, GL2 Mao, and Zhang, et al. 2016
thaliana (2016)
Arabidopsis Agro-transformation by floral dip AtU6 OST2 (AHA1) Osakabe et al. (2016) 2016
thaliana
Arabidopsis Protoplast transfection AtU6 EPSPS Sauer et al. (2016) 2016
thaliana
Arabidopsis Agro-transformation by floral dip AtU6 eIF(iso)4E Pyott, Sheehan, and 2016
thaliana Molnar (2016)
Arabidopsis Agro-transformation by floral dip AtU6 AtMIR169a, AtTFL1, AtMIR827 Zhao, Zhang, Liu, et al. 2016
thaliana (2016)
Arabidopsis Agrobacterium-mediated AtU3, PYR1, PYL1, PYL2, PYL4, PYL5, PYL8 Zhang et al. (2015) 2016
thaliana transformation AtU6,
At7SL
Arabidopsis na U3, U6 CBF1, CBF2 and CBF3, Zhao, Zhang, Xie, et al. 2016
thaliana (2016)
Arabidopsis Agro-transformation by floral dip AtU6 AGAMOUS, ELF6, REF6 SEP3, Yan, Chen, and Kaufmann 2016
thaliana At5g46910 (2016)
Arabidopsis Agro-transformation by floral dip AtU6 CLE18, GLV1,GLV2,GLV6, GLV7, Peterson et al. (2016) 2016
thaliana GLV8,GLV10
Arabidopsis Agro-transformation by floral dip AtU6 TRY, CPC, ETC2, CHLI1,CHLI2, Xie, Jones, Wang, Sun, and 2015
thaliana Zhang (2015)
Arabidopsis Agrobacterium-mediated AtU6 ADH1 Steinert, Schiml, Fauser, 2015
thaliana transformation and Puchta (2015)
Arabidopsis Agrobacterium-mediated AtU6 AtCRU Johnson, Gurevich, Filler, 2015
thaliana transformation Samach, and Levy (2015)
Arabidopsis Agrobacterium-mediated AtU6 Selected region Yuan et al. (2015) 2015
thaliana transformation
Arabidopsis Agrobacterium-mediated AtU6, AtCSTF64, miR159A, miR159B, Lowder et al. (2015) 2015
thaliana transformation AtU3 AtFIS2, miR319
Arabidopsis Agro-transformation by floral dip CaMV 35S ABP1 Gao, Zhang, et al. (2015) 2015
thaliana
Arabidopsis PEG-mediated protoplast BRI1, PHYB Woo et al. (2015) 2015
thaliana transfection
Arabidopsis Agrobacterium-mediated AtU6 FT, SPL 4 Hyun et al. (2015) 2015
thaliana transformation snRNA
Arabidopsis Agro-transformation by floral dip AtU6 NAC050, NAC052 Ning et al. (2015) 2015
thaliana
Arabidopsis Agro-transformation by floral dip AtU6 ADH1, TT4, RTEL1, GUUS, UGUS Fauser, Schiml, and Puchta 2014
thaliana and/or Stable Agro- (2014)
transformation
Arabidopsis Agro-transformation by floral dip AtU6 ADH1 Schiml et al. (2014) 2014
thaliana Stable Agro-transformation
Arabidopsis Agro-transformation by floral dip AtU6 Co-transfected GFP Jiang, Yang, and Weeks 2014
thaliana (2014)
Arabidopsis Agro-transformation by floral dip AtU6 BRI1, GAI, JAZ1,APA1,GUUS,TT4, Feng et al. (2014) 2014
(Continues)
DEMIRCI ET AL.
| 7

TABLE 2 (Continued)
gRNA
Species Delivery method promoter Target gene(s) Ref. Year
thaliana and/or Transient protoplast CHLI
transfection
Arabidopsis PEG Protoplast transfection and/ AtU6 PDS3, FLS2, RACK1b, RACK1c Li et al. (2013) 2013
thaliana or Leaf agroinfiltration
Arabidopsis Leaf agroinfiltration AtU6 Co-transfected GFP Jiang, Zhou, et al. (2013) 2013
thaliana
Arabidopsis Agro-transformation by floral dip AtU6 Co-transfected GUUS, TT4, CHLI1, Mao, Zhang, Xu, Zhang, 2013
thaliana and/or Stable Agro- CHL12 Gou, and Zhu (2013)
transformation
Arabidopsis Agro-transformation by floral dip AtU6 BRI1, GAI, JAZ1, YFFP Feng et al. (2013) 2013
thaliana and/or Transient protoplast
transfection
Brassica Agrobacterium-mediated AtU6 BolC.GA4.a Lawrenson et al. (2015) 2015
oleracea transformation
Citrus sinensis Agrobacterium-mediated CaMV35S CsLOB1 Jia, Orbovic, Jones, and 2016
transformation Wang (2015)
Citrus sinensis Agrobacterium-mediated leaf CaMV35S CsPDS Jia and Wang (2014a,b) 2014
agroinfiltration and/or Xcc-
facilitated leaf agroinfiltration
Cucumis Agro-transformation of AtU6 eIF4E Chandrasekaran et al. 2016
sativus cotyledone (2016)
Gycine max Stable Agro-transformation AtU6, GmPDS11, GmPDS18 Du et al. (2016) 2016
GmU6
Glycine max Agrobacterium rhizogenes- CaMV 35S Rj4 (Glyma.01G165800, Tang, Yang, Liu, and Zhu 2015
mediated hairy root Glyma.01G165800-D) (2015)
transformation
Glycine max A. rhizogenes-mediated hairy root AtU6 bar, GmFE12, GmSHR Cai et al. (2015) 2015
transformation
Glycine max A. rhizogenes-mediated MtU6 GFP, Glyma07g14530, Jacobs et al. (2015) 2015
transformation Particle Glyma01g38150 (01gDDM1),
bombardment of embryos Glyma11g07220 (11gDDM1),
miR1509, miR1514
Glycine max Particle bombardment GmU6 ALS1, DD20, DD43 Li et al. (2015) 2015
transformation
Glycine max A. rhizogenes-mediated hairy root AtU6 GS1, CHI20 Michno et al. (2015) 2015
transformation
Glycine max Agrobacterium-mediated GmU6, Glyma06g14180, Glyma08g02290 Sun et al. (2015) 2015
transformation PEG protoplast AtU6 and Glyma12g37050
transformation
Gossypium Agrobacterium-mediated AtU6 MYB 25-like Li et al. (2017) 2017
hirsutum transformation
Hordeum Agro-transformation of embryos TaU6 HvPM19 Lawrenson et al. (2015) 2015
vulgare
Lactuca sativa PEG-mediated protoplast DWD1, LsBIN2 Woo et al. (2015) 2015
transfection
Lotus Agro-transformation stable or LjU6 SYMRK, LjLb1, LjLb2, LjLb3 Wang, Wang, Tan, et al. 2016
japonicus hairy root (2016)
Marchantia Agro-transformation of sporelings MpU6 ARF1 Sugano et al. (2014) 2014
polymorpha
Medicago A. rhizogenes-mediated hairy root AtU6 GUS Michno et al. (2015) 2015
truncatula transformation
Nicotiana Agro-transformation by floral dip AtU6 NaAOC Kim et al. (2016) 2016
attenuata
Nicotiana PEG-mediated protoplast AOC Woo et al. (2015) 2015
attenuata transfection
(Continues)
8 | DEMIRCI ET AL.

TABLE 2 (Continued)
gRNA
Species Delivery method promoter Target gene(s) Ref. Year
Nicotiana Agro-inoculation of leaf U6 NbPDS Mubarik et al. (2016) 2016
benthamiana
Nicotiana Leaf agroinfiltration AtU6, XT Vazquez-Vilar et al. (2016) 2016
benthamiana OsU3

Nicotiana Agrobacterium-mediated AtU6 NbPDS3 and NbIspH Yin et al. (2015) 2015
benthamiana infiltration

Nicotiana Agrobacterium-mediated PEBV PDS Ali, Abul-faraj, Li, et al. 2015

benthamiana infiltration (2015); Ali, Abul-faraj,
Piatek, and Mahfouz
(2015)
Nicotiana Agrobacterium-mediated AtU6, NbFLS2, NbBAK1 Lowder et al. (2015) 2015
benthamiana infiltration AtU3

Nicotiana Leaf agroinfiltration AtU6 Bs3 promoter, PDS Piatek et al. (2015) 2015
benthamiana
Nicotiana Protoplast transfection and/or AtU6 NbPDS, NbPDS3 Li et al. (2013); Nekrasov 2013
benthamiana Leaf agroinfiltration and/or et al. (2013)
Transient protoplast
transfection
Nicotiana Leaf agroinfiltration AtU6 Co-transfected GFP Jiang, Zhou, et al. (2013) 2013
benthamiana
Nicotiana Leaf agroinfiltration CaMV35S PDS Upadhyay, Kumar, Alok, 2013
benthamiana and Tuli (2013)

Nicotiana Leaf agroinfiltration AtU6 PDS Alagoz et al. (2016) 2016

tabacum
Nicotiana Agrobacterium-mediated AtU6 NtPDS, NtFT4 Kaya, Mikami, Endo, Endo, 2016
tabacum transformation and Toki (2016)
Nicotiana Agro-transformation of callus U6 mCherry Mercx, Tollet, Magy, 2016
tabacum Navarre, and Boutry
(2016)
Nicotiana Protoplast transfection and/or AtU6 PDS, PDR6 Gao, Wang, et al. (2015) 2015
tabacum Agro-transformation of leaf
discs
Nicotiana Agrobacterium-mediated AtU6 ALS Baltes et al. (2014) 2014
tabacum infiltration
Oryza sativa Particle bombardment OsU3 OsALS Endo, Mikami, and Toki 2016
transformation and/or (2016); Sun et al. (2016)
Agrobacterium-mediated
transformation
Oryza sativa Particle bombardment of embryos OsU3 OsBEIIa Baysal et al. (2016) 2016
Oryza sativa Agrobacterium-mediated OsU6 Gn1a, DEP1, GS3, IPA1 Li, Li, et al. (2016) 2016
transformation
Oryza sativa Agrobacterium-mediated OsU3, sgRNA1-24 Liang, Zhang, Lou, and Yu 2016
transformation OsU6 (2016)
Oryza sativa Agrobacterium-mediated OsU6 OsDMC1, Mikami, Toki, and Endo 2016
transformation (2016)
Oryza sativa Agro-transformation of the OsU6 OsERF922 Wang, Wang, Liu, et al. 2016
embriyogenic calli (2016)
Oryza sativa Agrobacterium-mediated OsU6 OsROC5, OsDEP1 Zheng et al. (2016) 2016
transformation
Oryza sativa Agrobacterium-mediated OsU6 OsSPS genes Hashida et al. (2016); 2016
transformation Mikami, Toki, and Endo
(2015a)
Oryza sativa Protoplast transformation OsU3, OsEPSPS, OsDEP1 Li, Meng, et al. (2016) 2016
TaU3
(Continues)
DEMIRCI ET AL.
| 9

TABLE 2 (Continued)
gRNA
Species Delivery method promoter Target gene(s) Ref. Year
Oryza sativa Agrobacterium-mediated U3 OsPDS, Os02g23823, OsMPK2 Wang, Shen, Fu, Yan, and 2015
transformation Wang (2015)
Oryza sativa Agro-transformation of calli OsU6 gDL-1, Ikeda, Tanaka, Mikami, 2015
Endo, and Hirano (2015)
Oryza sativa Agro-transformation of mature OsU3 OsAOX1a, OsAOX1b, OsAOX1c, Xu et al. (2015) 2015
embriyonic calli OsBEL
Oryza sativa Agrobacterium-mediated OsU3 CDKA1, CDKA2, CDKB1, CDKB2 Endo, Mikami, and Toki, 2015
transformation 2014
Oryza sativa Protoplast transfection, Agro- OsU3, OsYSA, OsROC5 Lowder et al. (2015) 2015
transformation and/or OsU6
Agrobacterium-mediated
transformation
Oryza sativa Agrobacterium-mediated OsU3, YSA, PDS, DL, LigIV, ALS Mikami et al. (2015a) 2015
transformation OsU6
Oryza sativa Stable Agro-transformation OsRAV2 Duan et al. (2016) 2015
Oryza sativa PEG-mediated protoplast DWD1, P450 Woo et al. (2015) 2015
transfection
Oryza sativa Agrobacterium-mediated OsU3 DsRed, YSA, CDKB2 Mikami, Toki, and Endo 2015
transformation (2015b)
Oryza sativa Protoplast transfection Agro- OsU6 KO1, KOL5; CPS4, CYP99A2; Zhou et al. (2014) 2014
transformation of callus CYP76M5, CYP76M6, SWEET1a-
1b-11-13
Oryza sativa Agro-transformation of callus ZmU3 PDS, PMS3, EPSPS, DERF1, MSH1, Zhang et al. (2014) 2014
MYB5, MYB1, ROC5, SPP, YSA
Oryza sativa Agro-transformation of callus AtU6 BAL Zhang et al. (2014) 2014
Oryza sativa Protoplast transfection and/or OsU3 MPK2, Os02g23823, OsPDS, Gao et al. (2013) 2013
Particle bombardment of callus OsBADH2
Oryza sativa Protoplast transfection OsU3 or MPK5 Xie and Yang (2013) 2013
OsU6
Oryza sativa Protoplast transfection OsU6 SWEET14 Jiang, Zhou, et al. (2013) 2013
Oryza sativa Agro-transformation of callus OsU6 OsMYB1 Mao et al. (2013) 2013
Oryza sativa Agro-transformation of callus OsU3 CAO1, LAZY1, GUUS Miao et al. (2013) 2013
and/or Transient particle
bombardment of callus
Oryza sativa Agro-transformation of callus OsU6 ROC5, SPP, YSA Feng et al. (2013) 2013
Oryza sativa Transient protoplast transfection OsU3 OsPDS, OsBADH2 Gao et al. (2013) 2013
Oryza sativa PEG-mediated transformation and CSA Li, Zhang, et al. (2016) 2016
japonica Agrobacterium-mediated
transformation
Oryza sativa Agro-transformation of embryo- OsU6 CrRLK1L receptor kinase; RUPO Liu et al. (2016) 2016
japonica derived callus
Papaver Leaf agroinfiltration AtU6 40MT Alagoz et al. (2016) 2016
somniferum
Petunia Protoplast transfection PhNR locus Subburaj et al. (2016) 2016
hybrida
Petunia Agrobacterium-mediated AtU6 PhPDS Zhang, Yang, Yang, Li, and 2016
hybrida transformation Guo (2016)
Physcomitrella Protoplast transfection PpU6 PpAPT Collonnier et al. (2016) 2016
patens
Populus Agrobacterium-mediated AtU3, PDS Fan et al. (2015) 2015
tomentosa transformation AtU6
Populus Agrobacterium-mediated MtU6 4CL gene family Zhou et al. (2015) 2015
tremula transformation
Solanum Agrobacterium-mediated AtU6 SlPDS and SlPIF4 Pan et al. (2016) 2016
(Continues)
10 | DEMIRCI ET AL.

TABLE 2 (Continued)
gRNA
Species Delivery method promoter Target gene(s) Ref. Year
lycopersicum transformation
Solanum Agrobacterium-mediated NA SlBOP1, SlBOP2, SlBOP3, TFAM1, Xu et al. (2016) 2016
lycopersicum transformation TFAM2
Solanum Agrobacterium-mediated AtU6 ANT1 Čermák, Baltes, Čegan, 2015
lycopersicum transformation Zhang, and Voytas
(2015)
Solanum Agrobacterium-mediated AtU6 RIN Ito, Nishizawa-Yokoi, 2015
lycopersicum transformation Endo, Mikami, and Toki
(2015)
Solanum Agro-transformation of AtU6 SlAGO7, Solyc08g041770, Brooks, Nekrasov, 2014
lycopersicum cotyledons Solyc07g021170, Lippman, and Van Eck
Solyc12g044760 (2014)
Solanum Hairy root transformation by A. AtU6 SlSHR, SlSCR Ron et al. (2014) 2014
lycopersicum rhizogenes
Solanum Stable Agro-transformation AtU6 StALS1 Butler, Baltes, Voytas, and 2016
tuberosum Douches (2016)
Solanum PEG-mediated protoplast StU6, GBSS Andersson et al. (2016) 2016
tuberosum transfection AtU6
Solanum Agrobacterium-mediated StU6 StIAA2 Wang, Zhang, et al. (2015) 2015
tuberosum transformation
Solanum Agrobacterium-mediated AtU6 StALS1 Butler, Atkins, Voytas, and 2015
tuberosum infiltration Douches (2015)
Sorghum Agro-transformation of immature OsU6 Co-transfected DsRed Jiang, Zhou, et al. (2013) 2014
bicolor embryos
Taraxacum Stable Agro-transformation AtU6 1-FFT Iaffaldano et al. (2016) 2016
kok-saghyz
Triticum Particle bombardment of embryos TaU6 TaGASR7, TaDEP1, TaNAC2, Zhang, Liang, et al. (2016) 2016
aestivum TaPIN1, TaLOX2, TdGASR7,
TaGW2
Triticum Protoplast transfectionand/or TaU6 MLO-A1 Brooks et al. (2014); Wang 2014
aestivum Particle bombardment of et al. (2014)
embryos
Triticum Protoplast transfection TaU6 MLO Gao et al. (2013) 2013
aestivum
Triticum Agro-transfection of cells from CaMV35S PDS, INOX Upadhyay et al. (2013) 2013
aestivum immature embryos
Vitis vinifera Agrobacterium-mediated AtU6 IdnDH Ren et al. (2016) 2016
transformation
Zea mays Agrobacterium-mediated ZmU6 PSY1 Zhu et al. (2016) 2016
transformation
Zea mays Biolistic-mediated transformation ZmU6 ARGOS8 Shi et al. (2016) 2016
of immature embryos
Zea mays Particle bombardment of embryos ZmU6 LIG, MS26, MS45, ALS1, ALS2 Svitashev et al. (2015) 2015
Zea mays Protoplast transfection ZmU3 IPK Liang et al. (2014) 2014

also employed to generate large chromosomal deletions. In rice, large
chromosomal deletions (up to 115–245 kb) were observed, which
included three different clusters of genes (Zhou et al., 2014).
CRISPR/Cas system is also employed to modify non-coding
genome regions, including microRNAs (miRNAs) and long non-coding
RNAs (lncRNAs) in plants, which are important in plant growth and
development (Li & Zhang, 2016). Two soybean miRNA genes, miR1514
and miR1509, were targeted via CRISPR/Cas9 system and generated
indel frequencies were greater than 95% (Jacobs et al., 2015). Lowder
et al. (2015) employed CRISPR/Cas 9 to activate the expression of
miR319 gene and their results showed that expression of miR319
precursor was increased by 3- to 7.5-fold in Arabidopsis. Surprisingly,
they did not detected the expression level of final miRNA products and
did not report any phenotype of these genome-knockout lines. Zhao,
Zhang, Liu, et al. (2016) knocked out MIR169a and MIR827a with 20%
and 24% efficiency, respectively, in Arabidopsis; the mir169a knockout
DEMIRCI ET AL.
mutants exhibited greater drought tolerance than the wild-type plants
did.
Up to date, several studies employed CRISPR/Cas9 genome
editing tool to functionally analyze gene functions and several novel
traits were discovered in term of agricultural importance. In maize,
modifying a single native gene ARGOS8, a negative regulator of
ethylene responses, by CRISPR/Cas9 system showed increases in
maize grain yield under drought stress (Shi et al., 2016). In order to
improve Arabidopsis response to abiotic stress, Osakabe et al. (2016)
employed a truncated gRNA (tru-gRNA)/Cas9 system to generate
new alleles for OST2 gene, a proton pump in Arabidopsis with up to
32.8% average mutation rates. OST2 gene mutation changed the
pattern of the stomatal closing in response to environmental
conditions and enhanced plant tolerance to drought stress. A recent
study generated a herbicide-resistant rice through CRISPR/Cas9-
mediated homologous recombination of ALS1 (Acetolactate Synthase
1) (Sun et al., 2016). In a recent study, the ARF1 gene encoding auxin
response factor 1 was modified in a land plant Marchantia polymorpha
(Sugano et al., 2014). In opium poppy (Papaver somniferum L.), an
important medicinal aromatic plant, the 4′OMT2 gene which
regulates the biosynthesis of benzylisoquinoline alkaloids (BIAs)
was targeted to knock-out via nonhomologous end-joining genome
repair mechanism. InDels and HPLC-TOF/MS analyses showed that
efficient knocked-out in 4′OMT2 gene caused a significantly
accumulation of BIAs (e.g., morphine, thebaine) (Alagoz et al.,
2016). Another application of CRISPR/Cas9-mediated genome
editing on plants with limited genome information has been
performed to target the NR gene locus in Petunia hybrida (Subburaj
et al., 2016).
More and more evidences showed that CRISPR/Cas9 system is a
powerful tool to manipulate genetic elements in an individual genome;
it can be used to manipulated specific locus/region/sequence of DNA.
It is easy to design a sgRNA for CRISPR/Cas9 genome editing. With
whole-genome information, it is also easy to avoid the potential off-
target effects. This emerging technology will bring greatly contribution
to next-generation plant physiology and genetics research as well as
plant breeding.
ACKNOWLEDGMENTS
Authors kindly acknowledge Turkish Academy of Science (TUBA) with
grant type GEBIP and TUBITAK (113O016 to TU). We also appreciate
the partial support from the Cotton Incorporated (15–770 to BZ).
CONFLICT OF INTEREST
The authors have declared that no competing interests exist.
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