CRISPR-Cas9 An RNA-guided Highly Precise Synthetic Tool For Lant Genome Editing PDF

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, NC, USA
*Corresponding author:
Turgay Unver, PhD

International Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylül University, Balcova
35340 Izmir, Turkey
Phone: +90232 4126535
E-Mail: turgay.unver@deu.edu.tr
†This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/jcp.25970]
Received 18 April 2017; Revised 20 April 2017; Accepted 20 April 2017

Journal of Cellular Physiology
This article is protected by copyright. All rights reserved
DOI 10.1002/jcp.25970
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Abstract
CRISPR/Cas 9 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/Cas 9 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/Cas 9 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 and rice. CRISPR/Cas 9-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

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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 et al., 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 et al., 2016; Gratz et al., 2014; Hwang et al.,
2013a).
In 1987, CRISPR was discovered in Escherichia coli genome by Ishino and his colleagues while
identifying the iap gene product (Ishino et al., 1987). 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 et al., 1995). In 2000, a large
number of regularly spaced repeats with a related function were identified (Mojica et al., 2000).
These repetitive DNA sequences were only existed in prokaryotes but not in viruses and
eukaryotes, which were named as CRISPR (Jansen et al., 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 et al., 2005). In 2005, three independent research groups demonstrated that
some CRISPR spacers are originated from phage and plasmid DNA (Bolotin et al., 2005; Mojica
et al., 2005; Pourcel et al., 2005). However, until 2007, its function was still unclear. Barrangou
and his colleagues showed firstly an experimental evidence that CRISPR provides acquire
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resistance against a 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 and 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 betwwen
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 demonstrated in zebrafish (Hwang et al., 2013b) and bacterial cells (Jiang et al.,
2013a). 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 et al., 2013; Xie and Yang, 2013b).
Currently, a variety of plant species were modified by CRISPR/Cas9 technology, which include
Brassica oleracea (Lawrenson et al., 2015), Citrus sinensis (Jia and Wang, 2014a), Cucumis
sativus (Chandrasekaran et al., 2016), Glycine max (Jacobs et al., 2015), Gossypium hirsutum
(Li et al., 2017), Hordeum vulgare (Lawrenson et al., 2015), Lactuva sativa (Woo et al.,
2015), Lotus japonicus (Wang et al., 2016b), Marchantia polymorpha (Sugano et al., 2014),
Medicago truncatula (Michno et al., 2015), Nicotiana attenuate (Woo et al., 2015), N.
tabacum (Baltes et al., 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 et al., 2015), Solanum lycopersicum (Ron et al.,
2014), S. tuberosum (Wang et al., 2015b), Sorghum bicolor (Jiang et al., 2013b), Taraxacum

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kok-saghyz (Iaffaldano et al., 2016), Triticum aestivum (Gao et al., 2013) Vitis vinifera (Ren et
al., 2016) and Zea mays (Liang et al., 2014).
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 et al., 2011; Terns and Terns, 2011). Each
native 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 palindromic 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 et al., 2011b; Westra et al., 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 and 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 and 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 et al., 2009) and helps bacteria to protect

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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 et al., 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 et al., 2011a; Makarova et al., 2011b). 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 et al., 2011b). 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 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
(Fig.1A) (Rath et al., 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 (Fig.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 crRNA-guided DNA cleavage by Cas9 (Deltcheva et al., 2011). As reported by
Nishimasu and his colleagues, the crystal structure of S. pyogenes Cas9 (SpCas9), an RNA-
guided DNA endonuclease has two-lobed structure consisting of a target recognition (REC) lobe

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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 (Fig.1C) (Nishimasu et al., 2014). 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 recombination (HR, also called homology-directed repair,
HDR) (Fig.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 results 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 et al., 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 (Zhou et al., 2014). In a simple way, homologous recombination-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 et al., 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 (http://crispr.mit.edu). Some of them
offer available plant databases enabling to a design of sgRNAs and to explore possible off-target
sites (Stemmer et al., 2015) (Table 1). For example, CRISPR-PLANT
(http://www.genome.arizona.edu/crispr/) was established to help researchers for designing plant
genome editing CRISPR/Cas9 systems (Yang, 2014). Recently, an online web tool
(http://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

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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 and 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 monocotyledonous species can be used for sgRNA expression in only
corresponding plant clades being dicot or monocot plants (Mao et al., 2016a). 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 et al., 2013).
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. Selection 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 and 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 et al., 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 seven years
(Soundararaj et al., 1987). Although traditional methods have bred many new varieties, due to

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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 binding domain and a non-specific DNA cleavage module
from FokI, a type II restriction endonuclease (Kim et al., 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 and Kim, 2014). FokI restriction endonuclease is sit in
the C-terminus (Li et al., 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 et al., 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 and 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).

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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 methylation sensitivity, however, ZFNs and TALENs are sensitive to methylated DNA (Mao
et al., 2016a). 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.
Application of CRISPR/Cas in model and crop plant species

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 and 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

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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 et al., 2015a).
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 and Nakata,
2015). CRISPR/Cas9-mdiated gene replacement was achieved in the rice endogenous gene 5-
enolpyruvylshikimate-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 et al., 2016a). CRISPR/Cas9 genome editing is 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 and 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 and colleagues employed CRISPR/Cas 9 to activate the
expression of miR319 gene and their results show the expression of miR319 precursor was
increased by 3- to 7.5-fold in Arabidopsis (Lowder et al., 2015). Surprisingly, they didnot
detected the expression level of final miRNA products and didnot report any phenotype of these

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genome-knockout lines. Zhao and colleagues knocked out MIR169a and MIR827a with 20% and
24% efficiency, respectively, in Arabidopsis; the mir169a knockout mutants exhibited greater
drought tolerance than the wild-type plants did (Zhao et al., 2016b).
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 and
colleagues 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 (Osakabe et al., 2016). 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.

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Acknowledgements
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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23

Figure legend
Figure 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.
Table Legend
Table 1. Summary of available web tools for designing of CRISPR/Cas system.
Table 2. Applications of CRISPR/Cas9-mediated genome editing in plants.

24

Figure 1

25

Table 1. Summary of available web tools for designing of CRISPR/Cas system.
Name of the Software / Availability of Ref. Year

Web Site Link Plant
Databases
http://www.rgenome.net/cas-designer/ Yes (Park et al., 2016) 2016
CRISPR-DO
http://cistrome.org/crispr/ No (Ma et al., 2016) 2016
CRISPy No (Blin et al., 2016) 2016
http://crispy.secondarymetabolites.org/#/input
phytoCRISP-Ex
http://www.phytocrispex.biologie.ens.fr/CRISP-Ex/ No (Rastogi et al., 2016) 2016
CCTop - CRISPR/Cas9 target online predictor Yes (Stemmer et al., 2015) 2015
http://crispr.cos.uni-heidelberg.de/
http://stuparcrispr.cfans.umn.edu/CRISPR/ Yes (Michno et al., 2015) 2015
http://research.microsoft.com/en-us/projects/azimuth/ No (Fusi et al., 2015) 2015
CRISPRseek
http://www.bioconductor.org/packages/release/bioc/html/C
RISPRseek.html No (Zhu et al., 2014) 2014
CRISPR-PLANT Yes (Xie et al., 2014a) 2014
http://www.genome.arizona.edu/crispr/
SgRNAcas9
http://www.biootools.com/col.jsp?id=103/ No (Xie et al., 2014b) 2014
CasOT Yes (Xiao et al., 2014) 2014
http://eendb.zfgenetics.org/casot
(O'Brien and Bailey,
http://gt-scan.braembl.org.au Yes 2014) 2014
CHOPCHOP Yes (Montague et al., 2014
http://chopchop.cbu.uib.no/ 2014)
CRISPR-P
http://cbi.hzau.edu.cn/crispr Yes (Lei Y, 2014) 2014
E-CRISP Yes (Heigwer et al., 2014) 2014
http://www.e-crisp.org/E-CRISP/designcrispr.html
http://www.broadinstitute.org/rnai/public/analysis-
tools/sgrna-design No (Doench et al., 2014) 2014
Cas-OFFinder Yes (Bae et al., 2014) 2014
http://www.rgenome.net/cas-offinder/
CRISPRdirect
http://crispr.dbcls.jp/ Yes (Naito et al., 2014) 2014
CRISPR Design Tool Yes (Ran et al., 2013) 2013
http://crispr.mit.edu/ (Hsu et al., 2013)
http://www.genome-engineering.org

26

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 et al., 2016
thaliana transformation 2016)
At(MGE1p
Arabidopsis Agro-transformation by MGE2p, (Eid et al.,
thaliana floral dip MGE3p) ETC2, CPC, and TRY 2016) 2016
Arabidopsis Agro-transformation by AtU6 ABP1 (Gao et al., 2016
thaliana floral dip 2016)
Arabidopsis Agro-transformation by (Kim et al.,
thaliana floral dip AtU6 AtSH3P3 2016) 2016
Arabidopsis Agro-transformation by AtU6 AP1, TT4, GL2 (Mao et al., 2016
thaliana floral dip 2016b)
Arabidopsis Agro-transformation by (Osakabe et
thaliana floral dip AtU6 OST2 (AHA1) al., 2016) 2016
Arabidopsis Protoplast transfection AtU6 EPSPS (Sauer et al., 2016
thaliana 2016)
Arabidopsis Agro-transformation by (Pyott et al.,
thaliana floral dip AtU6 eIF(iso)4E 2016) 2016
Arabidopsis Agro-transformation by AtU6 AtMIR169a, AtTFL1, (Zhao et al., 2016
thaliana floral dip AtMIR827 2016b)
AtU3,
Arabidopsis Agrobacterium-mediated AtU6, PYR1, PYL1, PYL2, PYL4, (Zhang et al.,
thaliana transformation At7SL PYL5, PYL8 2015) 2016
Arabidopsis na U3, U6 CBF1, CBF2 and CBF3, (Zhao et al., 2016
thaliana 2016a)
Arabidopsis Agro-transformation by AGAMOUS, ELF6, REF6 (Yan et al.,
thaliana floral dip AtU6 SEP3, At5g46910 2016) 2016
Arabidopsis Agro-transformation by AtU6 CLE18, GLV1,GLV2,GLV6, (Peterson et 2016
thaliana floral dip GLV7,GLV8,GLV10 al., 2016)
Arabidopsis Agro-transformation by TRY, CPC, ETC2, (Wang et al.,
thaliana floral dip AtU6 CHLI1,CHLI2, 2015c) 2015
Arabidopsis Agrobacterium-mediated AtU6 ADH1 (Steinert et 2015
thaliana transformation al., 2015)
Arabidopsis Agrobacterium-mediated (Johnson et
thaliana transformation AtU6 AtCRU al., 2015) 2015
Arabidopsis Agrobacterium-mediated AtU6 Selected region (Yuan et al., 2015
thaliana transformation 2015)
Arabidopsis Agrobacterium-mediated AtCSTF64, miR159A, (Lowder et
thaliana transformation AtU6, AtU3 miR159B,AtFIS2, miR319 al., 2015) 2015
Arabidopsis Agro-transformation by CaMV 35S ABP1 (Gao et al., 2015
thaliana floral dip 2015b)
Arabidopsis PEG-mediated protoplast (Woo et al.,
thaliana transfection BRI1, PHYB 2015) 2015
Arabidopsis Agrobacterium-mediated AtU6 FT, SPL 4 (Hyun et al., 2015
thaliana transformation snRNA 2015)
Arabidopsis Agro-transformation by (Ning et al.,
thaliana floral dip AtU6 NAC050, NAC052 2015) 2015
Arabidopsis Agro-transformation by AtU6 ADH1, TT4, RTEL1, GUUS, (Fauser et al., 2014
thaliana floral dip and/or UGUS 2014)
Stable Agro-
27

transformation
Agro-transformation by
floral dip
Arabidopsis Stable Agro- (Schiml et al.,
thaliana transformation AtU6 ADH1 2014) 2014
Arabidopsis Agro-transformation by AtU6 Co-transfected GFP (Jiang et al., 2014
thaliana floral dip 2014)
floral dip and/or BRI1, GAI,
Arabidopsis Transient protoplast JAZ1,APA1,GUUS,TT4, (Feng et al.,
thaliana transfection AtU6 CHLI 2014) 2014
Arabidopsis PEG Protoplast AtU6 PDS3, FLS2, RACK1b, (Li et al., 2013
thaliana transfection and/or RACK1c 2013)
Leaf agroinfiltration
Arabidopsis (Jiang et al.,
thaliana Leaf agroinfiltration AtU6 Co-transfected GFP 2013b) 2013
Arabidopsis Agro-transformation by AtU6 Co-transfected GUUS ,TT4, (Mao et al., 2013
thaliana floral dip and/or CHLI1, CHL12 2013)
Stable Agro-
transformation
floral dip and/or
Arabidopsis Transient protoplast (Feng et al.,
thaliana transfection AtU6 BRI1, GAI, JAZ1, YFFP 2013) 2013
Brassica Agrobacterium-mediated AtU6 BolC.GA4.a (Lawrenson 2015
oleracea transformation et al., 2015)
Agrobacterium-mediated (Jia et al.,
Citrus sinensis transformation CaMV35S CsLOB1 2015) 2016
Citrus sinensis Agrobacterium-mediated CaMV35S CsPDS (Jia and 2014
leaf agroinfiltration and/or Wang,
Xcc-facilitated leaf 2014a)
agroinfiltration (Jia and
Wang,
2014b)
(Chandraseka
Agro-transformation of ran et al.,
Cucumis sativus cotyledone AtU6 eIF4E 2016) 2016
Gycine max Stable Agro- AtU6, GmPDS11, GmPDS18 (Du et al., 2016
transformation GmU6 2016)
Agrobacterium
rhizogenes-mediated hairy Rj4 (Glyma.01G165800, (Tang et al.,
Glycine max root transformation CaMV 35S Glyma.01G165800-D) 2015) 2015
Glycine max A. rhizogenes-mediated AtU6 bar, GmFE12, GmSHR (Cai et al., 2015
hairy root transformation 2015)
GFP, Glyma07g14530,
Glyma01g38150
A. rhizogenes-mediated (01gDDM1),
transformation Glyma11g07220
Particle bombardment of (11gDDM1),miR1509, (Jacobs et al.,
Glycine max embryos MtU6 miR1514 2015) 2015
Glycine max Particle bombardment GmU6 ALS1, DD20, DD43 (Li et al., 2015
transformation 2015)
A. rhizogenes-mediated (Michno et
Glycine max hairy root transformation AtU6 GS1, CHI20 al., 2015) 2015
Glycine max Agrobacterium-mediated GmU6, Glyma06g14180, (Sun et al., 2015
transformation AtU6 Glyma08g02290 and 2015)
28

PEG protoplast Glyma12g37050

transformation
Gossypium Agrobacterium-mediated (Li et al.,
hirsutum transformation AtU6 MYB 25-like 2017) 2017
Hordeum Agro-transformation of TaU6 HvPM19 (Lawrenson 2015
vulgare embryos et al., 2015)
PEG-mediated protoplast (Woo et al.,
Lactuca sativa transfection DWD1, LsBIN2 2015) 2015
Lotus japonicus Agro-transformation stable LjU6 SYMRK, LjLb1, LjLb2, (Wang et al., 2016
or hairy root LjLb3 2016b)
Marchantia Agro-transformation of (Sugano et
polymorpha sporelings MpU6 ARF1 al., 2014) 2014
Medicago A. rhizogenes-mediated AtU6 GUS (Michno et 2015
truncatula hairy root transformation al., 2015)
Nicotiana Agro-transformation by (Kim et al.,
attenuata floral dip AtU6 NaAOC 2016) 2016
Nicotiana PEG-mediated protoplast AOC (Woo et al., 2015
attenuata transfection 2015)
Nicotiana (Mubarik et
benthamiana Agro-inoculation of leaf U6 NbPDS al., 2016) 2016
Nicotiana Leaf agroinfiltration AtU6, OsU3 XT (Vazquez- 2016
benthamiana Vilar et al.,
2016)
Nicotiana Agrobacterium-mediated (Yin et al.,
benthamiana infiltration AtU6 NbPDS3 and NbIspH 2015) 2015
Nicotiana Agrobacterium-mediated PEBV PDS (Ali et al., 2015
benthamiana infiltration 2015a)
(Ali et al.,
2015b)
Nicotiana Agrobacterium-mediated (Lowder et
benthamiana infiltration AtU6, AtU3 NbFLS2, NbBAK1 al., 2015) 2015
Nicotiana Leaf agroinfiltration AtU6 Bs3 promoter, PDS (Piatek et al., 2015
benthamiana 2015)
Protoplast transfection
and/or
Leaf agroinfiltration (Li et al.,
and/or 2013)
Nicotiana Transient protoplast (Nekrasov et
benthamiana transfection AtU6 NbPDS, NbPDS3 al., 2013) 2013
Nicotiana Leaf agroinfiltration AtU6 Co-transfected GFP (Jiang et al., 2013
benthamiana 2013b)
Nicotiana (Upadhyay et
benthamiana Leaf agroinfiltration CaMV35S PDS al., 2013) 2013
Nicotiana Leaf agroinfiltration AtU6 PDS (Alagoz et 2016
tabacum al., 2016)
Nicotiana Agrobacterium-mediated (Kaya et al.,
tabacum transformation AtU6 NtPDS, NtFT4 2016) 2016
Nicotiana Agro-transformation of U6 mCherry (Mercx et al., 2016
tabacum callus 2016)
Protoplast transfection
and/or
Nicotiana Agro-transformation of (Gao et al.,
tabacum leaf discs AtU6 PDS, PDR6 2015a) 2015
Nicotiana Agrobacterium-mediated AtU6 ALS (Baltes et al., 2014
tabacum infiltration 2014)
Oryza sativa Particle bombardment OsU3 OsALS (Sun et al., 2016
29

transformation and/or 2016)

Agrobacterium-mediated (Endo et al.,
Oryza sativa Particle bombardment of OsU3 OsBEIIa (Baysal et al., 2016
embryos 2016)
Agrobacterium-mediated (Li et al.,
Oryza sativa transformation OsU6 Gn1a, DEP1, GS3, IPA1 2016b) 2016
Oryza sativa Agrobacterium-mediated OsU3, OsU6 sgRNA1-24 (Liang et al., 2016
Agrobacterium-mediated (Mikami et
Oryza sativa transformation OsU6 OsDMC1, al., 2016) 2016
Oryza sativa Agro-transformation of the OsU6 OsERF922 (Wang et al., 2016
embriyogenic calli 2016a)
Agrobacterium-mediated (Zheng et al.,
Oryza sativa transformation OsU6 OsROC5, OsDEP1 2016) 2016
Oryza sativa Agrobacterium-mediated OsU6 OsSPS genes (Hashida et 2016
transformation al., 2016)
(Mikami et
al., 2015a)
(Li et al.,
Oryza sativa Protoplast transformation OsU3, TaU3 OsEPSPS, OsDEP1 2016a) 2016
Oryza sativa Agrobacterium-mediated U3 OsPDS, (Wang et al., 2015
transformation Os02g23823, 2015a)
OsMPK2
Agro-transformation of (Ikeda et al.,
Oryza sativa calli OsU6 gDL-1, 2015) 2015
Oryza sativa Agro-transformation of OsU3 OsAOX1a, OsAOX1b, (Xu et al., 2015
mature embriyonic calli OsAOX1c, OsBEL 2015)
Agrobacterium-mediated CDKA1, CDKA2, CDKB1, (Endo et al.,
Oryza sativa transformation OsU3 CDKB2 2014) 2015
Oryza sativa Protoplast transfection, OsU3, OsU6 OsYSA, OsROC5 (Lowder et 2015
Agro-transformation al., 2015)
and/or Agrobacterium-
mediated transformation
Agrobacterium-mediated (Mikami et
Oryza sativa transformation OsU3, OsU6 YSA, PDS, DL, LigIV, ALS al., 2015a) 2015
Oryza sativa Stable Agro- OsRAV2 (Duan et al., 2015
PEG-mediated protoplast (Woo et al.,
Oryza sativa transfection DWD1, P450 2015) 2015
Oryza sativa Agrobacterium-mediated OsU3 DsRed, YSA, CDKB2 (Mikami et 2015
transformation al., 2015b)
KO1, KOL5; CPS4,
Protoplast transfection CYP99A2; CYP76M5,
Agro-transformation of CYP76M6, SWEET1a-1b- (Zhou et al.,
Oryza sativa callus OsU6 11-13 2014) 2014
Oryza sativa Agro-transformation of ZmU3 PDS, PMS3, EPSPS, (Zhang et al., 2014
callus DERF1, MSH1, MYB5, 2014)
MYB1, ROC5, SPP, YSA
Agro-transformation of (Zhang et al.,
Oryza sativa callus AtU6 BAL 2014) 2014
Oryza sativa Protoplast transfection OsU3 MPK2, Os02g23823, (Gao et al., 2013
and/or OsPDS, OsBADH2 2013)
Particle bombardment of
callus
Oryza sativa Protoplast transfection OsU3 or MPK5 (Xie and 2013
30

OsU6 Yang, 2013a)

Oryza sativa Protoplast transfection OsU6 SWEET14 (Jiang et al., 2013
2013b)
Agro-transformation of (Mao et al.,
Oryza sativa callus OsU6 OsMYB1 2013) 2013
Oryza sativa Agro-transformation of OsU3 CAO1, LAZY1, GUUS (Miao et al., 2013
callus and/or 2013)
Transient particle
bombardment of callus
Agro-transformation of (Feng et al.,
Oryza sativa callus OsU6 ROC5, SPP, YSA 2013) 2013
Oryza sativa Transient protoplast OsU3 OsPDS, OsBADH2 (Gao et al., 2013
transfection 2013)
PEG-mediated
transformation and
Oryza sativa Agrobacterium-mediated (Li et al.,
japonica transformation CSA 2016c) 2016
Oryza sativa Agro-transformation of OsU6 CrRLK1L receptor kinase; (Liu et al., 2016
japonica embryo-derived callus RUPO 2016)
Papaver (Alagoz et
somniferum Leaf agroinfiltration AtU6 40MT al., 2016) 2016
Petunia hybrida Protoplast transfection PhNR locus (Subburaj et 2016
al., 2016)
Agrobacterium-mediated (Zhang et al.,
Petunia hybrida transformation AtU6 PhPDS 2016a) 2016
Physcomitrella Protoplast transfection PpU6 PpAPT (Collonnier et 2016
patens al., 2016)
Populus Agrobacterium-mediated (Fan et al.,
tomentosa transformation AtU3, AtU6 PDS 2015) 2015
Populus tremula Agrobacterium-mediated MtU6 4CL gene family (Zhou et al., 2015
Solanum Agrobacterium-mediated (Pan et al.,
lycopersicum transformation AtU6 SlPDS and SlPIF4 2016) 2016
Solanum Agrobacterium-mediated NA SlBOP1, SlBOP2, SlBOP3, (Xu et al., 2016
lycopersicum transformation TFAM1, TFAM2 2016)
Solanum Agrobacterium-mediated (Čermák et
lycopersicum transformation AtU6 ANT1 al., 2015) 2015
Solanum Agrobacterium-mediated AtU6 RIN (Ito et al., 2015
lycopersicum transformation 2015)
SlAGO7, Solyc08g041770,
Solanum Agro-transformation of Solyc07g021170, (Brooks et
lycopersicum cotyledons AtU6 Solyc12g044760 al., 2014) 2014
Solanum Hairy root transformation AtU6 SlSHR, SlSCR (Ron et al., 2014
lycopersicum by A. rhizogenes 2014)
Solanum Stable Agro- (Butler et al.,
tuberosum transformation AtU6 StALS1 2016) 2016
Solanum PEG-mediated protoplast StU6, AtU6 GBSS (Andersson et 2016
tuberosum transfection al., 2016)
Solanum Agrobacterium-mediated (Wang et al.,
tuberosum transformation StU6 StIAA2 2015b) 2015
Solanum Agrobacterium-mediated AtU6 StALS1 (Butler et al., 2015
tuberosum infiltration 2015)
Agro-transformation of (Jiang et al.,
Sorghum bicolor immature embryos OsU6 Co-transfected DsRed 2013b) 2014
Taraxacum kok- Stable Agro- AtU6 1-FFT (Iaffaldano et 2016
saghyz transformation al., 2016)
31

TaGASR7, TaDEP1,
Triticum Particle bombardment of TaNAC2, TaPIN1, TaLOX2, (Zhang et al.,
aestivum embryos TaU6 TdGASR7, TaGW2 2016b) 2016
Triticum Protoplast TaU6 MLO-A1 (Wang et al., 2014
aestivum transfectionand/or 2014)
Particle bombardment of (Brooks et
embryos al., 2014)
Triticum (Gao et al.,
aestivum Protoplast transfection TaU6 MLO 2013) 2013
Triticum Agro-transfection of cells CaMV35S PDS, INOX (Upadhyay et 2013
aestivum from immature embryos al., 2013)
Agrobacterium-mediated (Ren et al.,
Vitis vinifera transformation AtU6 IdnDH 2016) 2016
Zea mays Agrobacterium-mediated ZmU6 PSY1 (Zhu et al., 2016
Biolistic-mediated
transformation of (Shi et al.,
Zea mays immature embryos ZmU6 ARGOS8 2016) 2016
Zea mays Particle bombardment of ZmU6 LIG, MS26, MS45, ALS1, (Svitashev et 2015
embryos ALS2 al., 2015)
(Liang et al.,
Zea mays Protoplast transfection ZmU3 IPK 2014) 2014

32

CRISPR-Cas9 An RNA-guided Highly Precise Synthetic Tool For Lant Genome Editing PDF

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CRISPR-Cas9 An RNA-guided Highly Precise Synthetic Tool For Lant Genome Editing PDF

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CRISPR/Cas9: an RNA-guided highly precise synthetic tool for plant genome

Yeliz Demirci1, Baohong Zhang2 , Turgay Unver1*

1 Izmir International Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylul

2 Department of Biology, East Carolina University, Greenville, NC, USA

Turgay Unver, PhD

Received 18 April 2017; Revised 20 April 2017; Accepted 20 April 2017

Keywords: CRISPR/Cas9; crop improvement; genome editing; plant breeding

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Origin of the CRISPR/Cas9 system

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Structural properties of CRISPR/Cas9 system

CRISPR-based defence mechanism consists of three phases: adaptation, expression, and

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Application of CRISPR/Cas in model and crop plant species

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Incorporated (15-770 to BZ).

The authors have declared that no competing interests exist.

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

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

Name of the Software / Availability of Ref. Year

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

PEG protoplast Glyma12g37050

transformation and/or 2016)

OsU6 Yang, 2013a)

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