Molecular Plant

Review Article

CRISPR/Cas9 Platforms for Genome Editing in

Plants: Developments and Applications
Xingliang Ma1,2,3, Qinlong Zhu1,2,3, Yuanling Chen1,2,3 and Yao-Guang Liu1,2,3,*
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China
2
Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, Guangzhou 510642, China
3
College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
*Correspondence: Yao-Guang Liu (ygliu@scau.edu.cn)
http://dx.doi.org/10.1016/j.molp.2016.04.009

ABSTRACT
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome
editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The
CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity
and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap-
tion of this technology to many plant species. In this review, we present an overview of current advances on
applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/
Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac-
tors affecting editing efficiency and specificity, and features of the induced mutations and applications of
the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9
technology and its significance for basic plant research and crop genetic improvement.
Key words: CRISPR/Cas9, genome editing, multiplex editing, mutation, plants
Ma X., Zhu Q., Chen Y., and Liu Y.-G. (2016). CRISPR/Cas9 Platforms for Genome Editing in Plants:
Developments and Applications. Mol. Plant. 9, 961–974.

INTRODUCTION regulation of gene expression, particularly for examination of

Genetic mutants are critical for the study of gene functions in
plants and for genetic improvement of crops. In the past de-
cades, characterization of natural mutants has revealed many
important biological mechanisms. Moreover, many studies
have used physical, chemical, or biological (e.g., T-DNA/
transposon insertion) mutagenesis to identify mutants and
construct mutant libraries corresponding to tens of thousands
of genes in model plants, such as Arabidopsis (Kuromori
et al., 2006) and rice (Wu et al., 2003; Yang et al., 2013).
However, random mutagenesis can produce many undesirable
mutations and rearrangements, and large-scale screening of
mutants remains tedious and costly (McCallum et al., 2000).
Other strategies, including antisense RNA (Mol et al., 1990),
virus-induced gene silencing (Baulcombe, 1999), and RNA
interference (Smith et al., 2000b), can interrupt the functions of
specific genes by repressing the corresponding mRNAs.
Although widely applied for gene functional studies in plants
(Waterhouse and Helliwell, 2003), the suppression occurs
indirectly, and often produces only a partial repressive effect.
Therefore, the output using these tools is not always
comparable with the use of genetic mutants. These methods
have provided useful insights, but basic plant research and
crop genetic improvement require new technologies for
targeted mutagenesis and precise editing of genes, and
the functions of members of gene families.
The emergence of programmable sequence-specific nucleases
(SSNs) provided a breakthrough in genome manipulation.
SSNs can induce double-stranded breaks (DSBs) in specific
chromosomal sites. The resulting DSBs can be repaired by
the error-prone non-homologous end joining (NHEJ) pathway,
often producing nucleotide insertions, deletions, and substitu-
tions. Another independent pathway, homology-directed repair
(HDR), also can repair the DSBs if homologous donor templates
are present at the time of DSB formation (Symington and
Gautier, 2011).
The zinc finger nucleases (ZFNs), as the first generation of SSNs
(Smith et al., 2000a; Bibikova et al., 2002), were used to edit plant
genomes (Lloyd et al., 2005; Zhang et al., 2010; Zhang and
Voytas, 2011; Petolino, 2015; Kumar et al., 2015). However,
ZFN constructs are difficult to manipulate and costly, which
greatly hinders their application in various organisms including
plants (Ramirez et al., 2008; Sanjana et al., 2012). Later, the
transcription activator-like effector nucleases (TALENs) adapted
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 9, 961–974, July 2016 ª The Author 2016. 961

Molecular Plant
Figure 1. Illustration of the Cas9/sgRNA Ribonuclease.
The Cas9 nuclease can be divided into two parts according to its structure
and function. The recognition part interacts with the sgRNA and target
DNA; the nuclease part has two nuclease domains (HNH and RuvC) and a
PAM (protospacer adjacent motif) interacting domain. The sgRNA is
produced by joining the crRNA and the transRNA with the artificial tetra-
loop (lower-case nucleotides). The target information is stored in the 50
terminal (in blue) of the sgRNA. The seed sequence (underlined in pink) of
the sgRNA is vital for the specificity of Cas9. Cas9 and the sgRNA form a
dual ribonuclease complex capable of binding the complementary strand
of the target site and creating a double-stranded break (DSB) three bases
upstream of the PAM.
from Xanthomonas bacteria were developed as a more promising
tool, and applied firstly in plants (Boch et al., 2009; Moscou and
Bogdanove, 2009; Boch and Bonas, 2010; Christian et al.,
2010; Li et al., 2011). Although easier to use than ZFNs,
TALENs still require construction of the complicated tandem
repeat domains in the TAL proteins. Recently, the clustered
regularly interspaced short palindromic repeat (CRISPR)-
associated protein9 (Cas9)-based genome editing tool,
CRISPR/Cas9, has been adapted from the type II CRISPR
adaptive immunity system in the bacterium Streptococcus
pyogenes (Jinek et al., 2012), and applied to genome editing in
many organisms including plants (Doudna and Charpentier,
2014). CRISPR/Cas9 can induce efficiently targeted mutations
based on base-pairing of the engineered single-guide RNAs
(sgRNAs) to the target DNA sites (Jinek et al., 2012); thus it is
much easier to manipulate than ZFNs and TALENs.
Several reviews have described the rapid development of the
CRISPR/Cas9 system for plants (Belhaj et al., 2015; Kumar and
Jain, 2015; Osakabe and Osakabe, 2015; Shan and Gao, 2015;
Weeks et al., 2015). Here we present an overview of recent
advances on development and application of CRISPR/Cas9
system in plants, emphasizing the general technical issues for
the establishment of plant CRISPR/Cas9 platforms. In addition,
we provide a perspective on future development and
challenges of the CRISPR/Cas technology and its significance
for basic plant research and crop genetic improvement.
STRUCTURE AND CLEAVAGE ACTIVITY
OF THE CAS9 NUCLEASE
CRISPR/Cas9 technology involves three major components: the
Cas9 protein, sgRNA, and target sites directly upstream of a pro-
tospacer adjacent motif (PAM; NGG for SpCas9 from S. pyo-
genes) (Jinek et al., 2012; Doudna and Charpentier, 2014). The
962 Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
CRISPR/Cas9 Platforms for Plant Genome Editing
artificial sgRNA is engineered by linking the DNA sequences
that express the protospacer-containing CRISPR RNA (crRNA)
and the trans-activating crRNA (tracrRNA); the 50 terminal part
of the sgRNA contains the target sequence (not including PAM)
for its pairing to the target site (Jinek et al., 2012; Cong et al.,
2013; Mali et al., 2013). The sgRNA accurately mimics
the original crRNA–tracrRNA duplex and greatly simplifies
manipulation with the CRISPR/Cas9 system. The Cas9 protein
and the sgRNA form a Cas9 nuclease complex (Jinek et al.,
2012; Jiang et al., 2015). Crucially, the specific recognition of
the Cas9/sgRNA nuclease complex to target sites can be
designed simply by introducing the target sequences into the
sgRNA (Jinek et al., 2012). This feature distinguishes the
CRISPR/Cas9 system from TALENs and ZFNs, which depend
on the DNA binding domains in the proteins for target
recognition. Both in vitro and in vivo, the Cas9/sgRNA complex
can search the genomic target DNA sequence adjacent to a
PAM, which is required for effective target recognition
(Sternberg et al., 2014). Once the target site is located, Cas9
melts the target sequence so that the sgRNA can pair with the
target complementary strand. The RucV and HNH domains of
Cas9 then cut both strands of the target DNA three bases
upstream of PAM, producing a blunt-ended DSB (Figure 1).
DSB can be repaired either by NHEJ to produce mutations in
the targeted site or by HDR in the presence of a homologous
donor DNA template to produce gene (fragment) knockin/
replacement for precise gene editing. Importantly, multiple
engineered sgRNAs with different target sequences can direct
Cas9 to the corresponding sites in the same cells (Cong et al.,
2013). Multiplex editing in the same cells or individuals has
many potential applications, such as mutation of functionally
related genes that control complex traits as well as multiple
members of gene families (Xie et al., 2015; Ma et al., 2015b).
CRISPR/CAS9 VECTOR SYSTEMS FOR
PLANTS
Upon the establishment of the CRISPR/Cas9 system in human
cells (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013), the
application of this technology was successfully tested in plant
cells, although the editing efficiencies in these cases were
relatively low (Li et al., 2013; Nekrasov et al., 2013; Shan et al.,
2013). Shortly thereafter, various CRISPR/Cas9 vector systems
were developed for more efficient editing of plant genomes,
including gene knockout, genomic deletion, disruption of cis-
regulatory elements, gene knockin, and suppression of virus
infection (Tables 1 and 2).
sgRNA Expression Cassettes
A typical sgRNA contains 98 nucleotides (including a 20-nt target
sequence) and functions as a guide in the Cas9/sgRNA nuclease
complex (Nishimasu et al., 2014). The expression of sgRNAs in
plants are generally driven by U3 or U6 small nuclear RNA gene
promoters, and sgRNAs are transcribed by RNA polymerase III
(Jiang et al., 2013; Li et al., 2013; Nekrasov et al., 2013; Shan
et al., 2013). Since the U3/U6-promoter:sgRNA expression cas-
settes are small (300–600 bp), target-adaptor ligation or over-
lapping PCR can be used to generate these sgRNA expression
cassettes with target sequences (Li et al., 2013; Mao et al.,
2013; Shan et al., 2013; Xie and Yang, 2013; Ma et al., 2015b)

Tested plant Promoter of Cas9; codon
species optimization of Cas9
Oryza sativa 2 3 P35S; rice
OsUbi (from rice); human
OsUbi; rice
2 3 P35S; plant
OsUbi (from rice)
ZmUbi (from maize); rice
Arabidopsis AtUbi (from Arabidopsis);
thaliana human
PcUbi (from Petroselinum
crispum); Arabidopsis
2 3 P35S; maize
ICU2 (from Arabidopsis); rice
Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
Maximum co-editing
targets; transformation
Promoter of sgRNA method
OsU3 1; Particle bombardment/
callus
OsU3 2; Agrobacterium/callus
OsU6.1, OsU6.2 4; Agrobacterium/callus
AtU6-26 1; Agrobacterium/callus
OsU3 8; Agrobacterium/callus
OsU3, OsU6a, OsU6b, 8; Agrobacterium/callus
OsU6c
AtU6 2; Agrobacterium/floral dip
AtU6-26 1; Agrobacterium/floral dip
AtU6-26, AtU6-29 2; Agrobacterium/floral dip
AtU6 4; Agrobacterium/floral dip
CRISPR/Cas9 Platforms for Plant Genome Editing
Mutant efficiency; main
Multiplex strategy types of mutants References
– 8.2% (16/194); biallelic, Shan et al., 2013
homozygous, heterozygous
Sequential cloning 45.3% (225/491); biallelic, Zhang et al., 2014
homozygous, heterozygous,
chimeric
Sequential cloning 67.9% (19/28); biallelic, Zhou et al., 2014a
homozygous, heterozygous
- 8.6% (18/184) Xu et al., 2014
Polycistronic tRNA Up to 100% Xie et al., 2015
process system
Golden Gate/Gibson 85.4% (280/328); biallelic, Ma et al., 2015b
assembly homozygous, heterozygous
Sequential cloning 81.2% (190/234); chimeric Mao et al., 2013;
Feng et al., 2014
– 26.7%; chimeric Fauser et al., 2014
Golden Gate/Gibson 82.6%; chimeric Xing et al., 2014
assembly
Sequential cloning 20.6%; chimeric Hyun et al., 2015

2 3 P35S; rice AtU3b, AtU3d, AtU6-1, 4; Agrobacterium/floral dip Golden Gate/Gibson 35.6%; chimeric Ma et al., 2015b
AtU6-29 assembly
EC1.2 (from Arabidopsis); AtU6-26, AtU6-29 2; Agrobacterium/floral dip – –; homozygous, biallelic, Wang et al., 2015b
Arabidopsis chimeric;
YAO (from Arabidopsis) AtU6-26 1; Agrobacterium/floral dip Sequential cloning 88.5%–90.5%; chimeric Yan et al., 2015
AtUbi AtU6, AtU3b, At7SL 6; Agrobacterium/floral dip Multiple enzymes/ 13%–93%; – Zhang et al., 2015
sequential cloning
Nicotiana P35S; human AtU6 1; Agroinfiltration/leaf – 6.7% (2/30); chimeric Nekrasov et al.,
benthamiana tissue 2013
2 3 P35S; tobacco AtU6-26 2; Agrobacterium/leaf – 81.8%–87.5%; chimeric Gao et al., 2015b
tissue
Solanum P35S; human AtU6 2; Agrobacterium/ Golden Gate 48% (14/29) (dual targets); Brooks et al., 2014
lycopersicum cotyledon segments homozygous, biallelic, chimeric

Molecular Plant
Zea mays ZmUbi (from maize); maize OsU3, TaU3 2; Agrobacterium/immature Golden Gate/Gibson 60% (dual targets); biallelic, Xing et al., 2014
embryos assembly homozygous
ZmUbi (from maize); maize ZmU6 3; Particle bombardment/ Co-delivery 77%–100%; biallelic, Svitashev et al.,
immature embryos heterozygous 2015

Table 1. Summary of Plant CRISPR/Cas9 Vector Platforms Designed for Stable Transformation.
963

(Continued on next page)

Molecular Plant CRISPR/Cas9 Platforms for Plant Genome Editing
(Figure 2). Ma et al. (2015b) designed a PCR-based, intermediate
cloning-free strategy to rapidly prepare sgRNA expression cas-

Wang et al., 2015a

Wang et al., 2014
settes; these cassettes are directly cloned into the CRISPR/

Lawrenson et al.,

Lawrenson et al.,
Zhou et al., 2015

Fan et al., 2015

Cas9 binary vectors by Golden Gate cloning or Gibson assembly

Li et al., 2015
References

(see below). Another strategy uses the ribozyme system to pro-

duce functional sgRNA by processing a pre-RNA transcribed

2015

2015
by RNA polymerase II, whereby regular tissue-specific or induc-
ible promoters can be used to express sgRNAs for special uses
(Gao and Zhao, 2014). The polycistronic tRNA processing
system also was used to generate sgRNA units by flanking the
sgRNAs with tRNA precursor sequences (Xie et al., 2015).
Mutant efficiency; main

Cas9 Expression Cassettes

types of mutants

The original coding sequence of Cas9 is 4107 bp in length. In eu-

50.9% (30/59)
5.6% (4/72); –

karyotes, nuclear localization of Cas9 requires fusion of a single

83.3% (5/6)

10%–23%

59%–76%
or dual nuclear localization signal (NLS) to the Cas9 coding
sequence. To improve Cas9 expression in plants, most modified
10%

Cas9 genes for plant genome editing have also been optimized
–

with plant-usage bias codons (Jiang et al., 2013; Li et al., 2013;

Miao et al., 2013; Shan et al., 2013; Fauser et al., 2014; Xing
Multiplex strategy

et al., 2014; Zhou et al., 2014a; Svitashev et al., 2015; Gao

et al., 2015a; Ma et al., 2015b). For use in rice and other
Golden Gate

Golden Gate

Gramineae, the codon-optimized Cas9 gene (Cas9p) with a

high editing efficiency has been further modified by increasing
the GC contents in the 50 terminal region (Ma et al., 2015b),
which mimics Gramineae genes (Wong et al., 2002). However,
–

several applications in plants have also used Cas9 codon-

4; Agrobacterium/leaf discs

1; Agrobacterium/immature

optimized for non-plant species, but their editing efficiency may

1; Particle bombardment/

1; particle bombardment/
targets; transformation

1; Agrobacterium/callus

be relatively lower than those of the plant-optimized methods

1; Agrobacterium/stem
Maximum co-editing

cotyledonary petioles

(Jiang et al., 2013; Mao et al., 2013; Nekrasov et al., 2013; Xie
immature embryos

2; Agrobacterium/

and Yang, 2013; Lawrenson et al., 2015).

In general, constitutive promoters, such as those of Ubiquitin

segments

embryos

gene of maize, rice, and Arabidopsis, and Cauliflower mosaic vi-

method

callus

rus (CaMV) 35S, can fulfill the requirement for driving the Cas9
gene in monocot and dicot plants to mediate efficient genome
editing in callus-based transformation methods. In many cases
AtU3b, AtU3d, AtU6-1,

the stronger Ubiquitin promoters produced higher editing effi-

U6-9-1 from soybean
Promoter of sgRNA

U6.6 from Medicago

TaU6 (from Triticum

ciencies than the CaMV 35S promoter (Table 1). However, use
of other promoters with high activity in the germline cell or
dividing tissue is more desirable for driving Cas9 toward
truncatula

improving the editing efficiency in Arabidopsis (see below).

aestivum)
AtU6-29

AtU6-26
TaU6

StU6

Delivery of the Cas9 and sgRNA Expression Cassettes

into Plant Cells
To mediate genome editing in vivo, vector constructs carrying the
Promoter of Cas9; codon

Cas9 and sgRNA expression cassettes must be delivered into the

optimization of Cas9

plant cells. To verify the feasibility and efficiency of the CRISPR/

Cas9 system in the early stages of testing, researchers generally
2 3 P35S; human

CsVMW; humans

EF1A2; soybean
2 3 P35S; plant

ZmUbi; humans

used a transient expression system to directly deliver plasmids

2 3 P35S; rice
ZmUbi; plant

carrying the Cas9 and sgRNA expression cassettes into proto-

plasts or employed vacuum infiltration of tobacco or Arabidopsis
leaves (Jiang et al., 2013). The Cas9 cassettes and sgRNA
cassettes can be arranged in separate constructs or single
Table 1. Continued

constructs (Jiang et al., 2013; Li et al., 2013; Nekrasov et al.,

Tested plant

2013; Shan et al., 2013; Xie and Yang, 2013). However, in many
Glycine max
tuberosum

tomentosa

plant species it is difficult to obtain transgenic plants from

Hordeum
aestivum
Solanum

oleracea
Brassica
species

Populus
Triticum

vulgare

protoplasts with heritable targeted mutations, thus limiting the

utility of the protoplast system. A recent study also reported a
DNA-free strategy for plant genome editing by transfecting
964 Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
 CRISPR/Cas9 Platforms for Plant Genome Editing
Plant species Target gene Function Knockout/-in, phenotype References
O. sativa CAO1 Synthesis of chlorophyll b Knockout, pale-green leaf Miao et al., 2013
LAZY1 Regulate shoot gravitropism, control Knockout, pronounced tiller spreading Miao et al., 2013
tiller angle
OsPDS Encoding phytoene desaturase Knockout, albino and dwarf Shan et al., 2013
Promoter region of OsSWEET Plant disease susceptibility genes Modification of the promoter, plant Zhou et al., 2014a
disease resistance
OsWaxy Amylose synthesis Knockout, glutinous rice Ma et al., 2015b
ALS Encoding acetolactate synthase Knockin, resistance to sulfonylurea Endo et al., 2016
involved in branched amino acid herbicides, gene replacement
biosynthesis
ALS Encoding acetolactate synthase Knockin, resistance to sulfonylurea Sun et al., 2016
involved in branched amino acid herbicides
biosynthesis
A. thaliana CHL1 The magnesium chelatase subunit I Knockout, albino (double mutant); pale Mao et al., 2013
CHL2 genes green (either mutant)
ABP1 Vital for perception of auxin and Knockout, no identifiable phenotype Gao et al., 2015b
developmental processes
ADH1 Turns allyl alcohol to highly toxic Knockout, survival after allyl alcohol Fauser et al., 2014
acrylaldehyde treatment
NPTII Kanamycin resistance cassette Knockin, kanamycin resistance Schiml et al., 2014
Beet severe curly top virus Virus infection Suppression, virus resistance Ji et al., 2015
Molecular Plant 9, 961–974, July 2016 ª The Author 2016.

TFL1 – Knockin, eGFP expression Zhao et al., 2016

N. benthamiana NtPds Phytoene desaturase Knockout, albino Gao et al., 2015a
NtPDR6 One ABC transporter participating in Knockout, more branches Gao et al., 2015a
strigolactone transport
Bean yellow dwarf virus (BeYDV) Virus infection Suppression, virus resistance Baltes et al., 2015
Tomato yellow leaf curl virus (TYLCV); Virus infection Suppression, virus resistance Ali et al., 2015b
Beet curly top virus (BCTV); (transient expression)
Merremia mosaic virus (MeMV)
S. lycopersicum SlAGO7 Biogenesis of trans-acting short Knockout, needle-like or wiry leaves Brooks et al., 2014
interfering RNAs
T. aestivum TaMLO homologs Repress the resistance pathway to Knockout, resistance to powdery Wang et al., 2014
powdery mildew mildew

Molecular Plant
H. vulgare HvPM19 ABA-inducible plasma membrane Knockout, related to grain dormancy Lawrenson et al., 2015
protein
B. oleracea BolC.GA4. Ortholog of Arabidopsis GA4a Knockout, dwarf phenotype Lawrenson et al., 2015

Table 2. Applications of CRISPR/Cas9-Based Editing of Genes in Plants.

(Continued on next page)
965
Molecular Plant CRISPR/Cas9 Platforms for Plant Genome Editing
preassembled complexes of purified Cas9 protein and synthe-
sized sgRNA into plant protoplasts (Woo et al., 2015). This
Svitashev et al., 2015
strategy is particularly useful for vegetatively propagated plants

Cermak et al., 2015

to generate transgene-free genome-edited lines.
Liang et al., 2014

Zhou et al., 2015

Zhou et al., 2015
Fan et al., 2015

Li et al., 2015
References

To obtain transgenic plants carrying targeted mutations, biolistic

transformation of calli and immature embryos can be used to
integrate the Cas9 and sgRNA expression constructs into plant
genomes and produce heritable mutations (Shan et al., 2013).
Biolistic transformation can also deliver in vitro synthesized
sgRNA directly into Cas9-transformed plant cells and induce
Knockout, condensed tannins content

targeted mutations (Svitashev et al., 2015).

Knockin, resistance to sulfonylurea

Knockin, resistance to sulfonylurea

Knockin, resistance to hygromycin
Knockout, reduce in lignin accrual

Since the Agrobacterium-mediated transformation method is the

herbicides, gene replacement

herbicides, gene replacement

most effective for many plants, most applications of CRISPR/
Knockout/-in, phenotype

Knockin, ectopic pigment Cas9 in plants use this method to integrate T-DNAs carrying both
the Cas9 and sgRNA expression cassettes into the plant
genomes (Table 1). Genome editing of Arabidopsis generally
Knockout, albino

uses Agrobacterium-mediated floral dip transformation (Table 1).

accumulation

In contrast, for other monocot and dicot plants, such as rice,

maize, tobacco, tomato, potato, and poplar, CRISPR/Cas9-
reduction

based genome editing approaches generally use Agrobacterium-

mediated stable transformation of callus, immature embryos, or
–

other tissues (Table 1). In addition, the agroinfiltration approach

has been exploited to introduce the Tobacco rattle virus DNA
carrying an sgRNA expression cassette into Cas9-transformed
Antinutritional phytic acid biosynthesis

tobacco (Yin et al., 2015; Ali et al., 2015a).

Hygromycin phosphotransferase
involved in branched amino acid

involved in branched amino acid

Encoding acetolactate synthase

Encoding acetolactate synthase

STRATEGIES FOR MULTIPLEX GENOME

Anthocyanin biosynthesis
Flavonoid biosynthesis

TARGETING IN PLANTS
Phytoene desaturase
Wood discoloration

Simultaneous editing of multiple genomic sites in plants has

many applications, such as studying multiple related genes or
biosynthesis

biosynthesis

knockout of functionally redundant genes, or genetic improve-

Function

ment of multiple traits in crop breeding. As already mentioned,

CRISPR/Cas9-based genome editing mostly depends on Agro-
bacterium-mediated transformation to obtain stable transgenic
plants. However, co-transforming multiple T-DNAs from separate
binary vectors into plant cells by Agrobacterium is tricky and un-
controllable (Hiei and Komari, 2008). Therefore, several strategies
have been developed to assemble multiple sgRNA expression
ZmIPK1A, ZmIPK and ZmMRP4

cassettes into single CRISPR/Cas9 binary constructs.

Sequential rounds of regular cloning can insert a few sgRNA

expression cassettes containing different targets into a single bi-
nary vector (Li et al., 2013; Mao et al., 2013; Zhou et al., 2014a; Yan
et al., 2015). Alternatively, multiple restriction enzymes that yield
Target gene

DD43 region

sequential compatible palindromic sticky ends can be used

to insert a few (usually up to three) sgRNA cassettes into a
potPDS

ANT1

vector (Zhang et al., 2015; Wang et al., 2015b) (Figure 2A).

ALS2

ALS1
4CL1
4CL2

This traditional cloning method can clone only few sgRNA

expression cassettes into the CRISPR/Cas9 binary vectors and/
or needs multiple rounds of cloning, and thus is time-consuming.
Populus trichocarpa

Table 2. Continued

The Golden Gate cloning method uses distinctive type

S. lycopersicum
Plant species

II restriction enzymes, such as BsaI, to generate sequential

compatible, non-palindromic sticky ends among multiple DNA
Z. mays

G. max

fragments; thus Golden Gate cloning can simultaneously and effi-

ciently ligate multiple DNA fragments in a given arrangement
(Engler et al., 2008). Based on this cloning strategy, two sets of
966 Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
CRISPR/Cas9 Platforms for Plant Genome Editing Molecular Plant

Figure 2. Strategies for Generation of Multiple sgRNA Expression Cassettes in a Binary Vector.
(A) Cloning of multiple sgRNA expression cassettes using multiple restriction enzymes (RE1–RE4). The sgRNA expression cassettes driven by U3/U6
promoters (Pr) are prepared in an intermediate vector, and recovered by digestion with the two corresponding restriction enzymes. A few (usually up to
three) cassettes can be ligated simultaneously to a binary vector. Since the palindromic sticky ends cause competitive self-ligation, simultaneous cloning
of more than three fragments to a vector is difficult.
(B) Assembly of multiple (three as an example) sgRNA expression cassettes using Golden Gate ligation. The sgRNA expression cassettes are prepared
in vitro by PCR amplification of the ligated cassettes. The cassettes are digested with a type II restriction endonuclease (BsaI) to produce non-palindromic
sticky ends, and ligated simultaneously to a binary vector for cloning.
(C) Cloning multiple (three as an example) sgRNA expression cassettes using Gibson assembly. The sgRNA expression cassettes with homologous
region ends (HR1–HR4) are prepared in vitro by PCR and ligated to a binary vector in a Gibson assembly reaction.
(D) Generation of multiple sgRNAs by the polycistronic tRNA-gRNA gene. Multiple (three as an example) pre-tRNA/sgRNA scaffolds are linked to a U3/U6
promoter by Golden Gate ligation and cloned into an intermediate vector and then cloned into a binary vector. In the transgenic plants, the transcribed
polycistronic pre-tRNA/sgRNA is processed (red arrows) in vivo to release independent sgRNAs with different target sequences.

CRISPR/Cas9 vector systems have been developed to prepare
CRISPR/Cas9 binary constructs with multiple PCR-prepared
sgRNA expression cassettes in a single round of cloning (Xing
et al., 2014; Ma et al., 2015b) (Figure 2B). Another CRISPR/
Cas9 vector uses an alternative strategy for sgRNA cassette
construction and assembly by preparing multiple single sgRNA
cassettes in intermediate vectors, digesting and recovering the
sgRNA cassettes, then cloning into a binary vector using
Golden Gate ligation (Lowder et al., 2015). With Golden Gate
cloning CRISPR/Cas9 binary constructs with multiple cassettes
(up to eight) were prepared, and seven FT-like genes were
simultaneously mutated in the same rice plants (Ma et al., 2015b).
The Gibson assembly method can efficiently join multiple DNA
fragments with homologous termini using the concerted actions
of the T5 exonuclease, Phusion DNA polymerase, and Taq DNA
ligase (Gibson et al., 2009). Similarly, this approach was used
to assemble multiple sgRNA expression cassettes in single
reactions into the CRISPR/Cas9 binary vectors (Ma et al.,
2015b) (Figure 2C).
The polycistronic tRNA-gRNA (PTG) system has been explored
to generate multiple sgRNAs with different target sequences
by flanking the sgRNAs with a tRNA precursor sequence (pre-
RNA). Upon transcription driven by a U3 or U6 promoter in
Molecular Plant 9, 961–974, July 2016 ª The Author 2016. 967
Molecular Plant
transgenic plants, in vivo processing of the pre-tRNAs releases
the sgRNAs from the polycistronic pre-RNA for multiplex genome
targeting (Xie et al., 2015) (Figure 2D). However, the upper limit of
the number of the polycistronic pre-tRNA/sgRNA that can be
driven by a U3 or U6 promoter remains unclear.
ANALYSIS OF TARGETED MUTATIONS
A first step in validating a newly established CRISPR/Cas9 vector
system, or applying the established CRISPR/Cas9 system to a
new plant species, should be to determine the editing efficiency.
In addition, the genotype of the resultant mutants must be deter-
mined before carrying out further studies. This requires detection
of the targeted mutations and sequencing of the mutated sites.
Using Reporter Genes to Verify Targeted Mutations
To rapidly verify the function of a newly established CRISPR/
Cas9 vector system, reporter genes, such as those encoding
b-glucuronidase or a fluorescent protein (GFP, YFP, or RFP),
can be used as an indicator of editing events. For example, the
reporter gene can be designed to contain a target site that causes
a frameshift; mutation of the target by the Cas9/sgRNA complex
may correct the reading frame of the gene to restore function
(Jiang et al., 2013; Feng et al., 2014). Alternatively, the reporter
gene can be designed to contain a duplicated region; a Cas9-
induced DSB can produce recombination between the dupli-
cated regions to restore the function of the reporter gene through
the single-strand annealing DNA repair (Siebert and Puchta,
2002; Mao et al., 2013).
Using Endonucleases to Verify Targeted Mutations
If the Cas9/sgRNA cleavage of the target is designed to contain a
restriction enzyme site, the targeted mutations may destroy the
restriction enzyme site; thus, restriction cutting before or after
PCR amplification can enrich mutated sequences (Lloyd et al.,
2005; Voytas, 2013). Several studies have exploited this
method to determine the presence of targeted mutations and
measure the editing efficiency (Jiang et al., 2013; Nekrasov
et al., 2013; Shan et al., 2013; Xie and Yang, 2013). However,
this strategy restricts target sequences to those that contain a
restriction enzyme site.
The Surveyor nuclease and T7 Endonuclease I assays can cleave
hybrid DNA fragments in PCR amplicons if the DNA duplex con-
tains unpaired nucleotides. Some studies, therefore, used this
method to detect the targeted mutations and determine the
mutagenesis efficiency (Guschin et al., 2010). This method is
suitable for any target sequence, but the detection sensitivity is
lower than that of the restriction enzyme site-based method
(Voytas, 2013).
Using PAGE to Verify Targeted Mutations
Single-strand DNAs with nucleotide variations can change their
conformations and exhibit differential migration rates in a non-
denaturing PAGE gel, so-called single-strand conformation poly-
morphism (SSCP). This SSCP method was used to detect muta-
tions induced by CRISPR/Cas9 (Zheng et al., 2016). Another
PAGE-based method also was used for detection of heterodu-
plex DNAs with targeted mutations (Zhu et al., 2014).
968 Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
CRISPR/Cas9 Platforms for Plant Genome Editing
Using High-Resolution Melting to Verify Targeted
Mutations
PCR amplicons with and without a mutation may differ in their
melting temperature, which can be detected by a high-
resolution melting assay. Thus, this method can also be used to
screen targeted mutations (Dahlem et al., 2012; Fauser et al.,
2014), but the detection sensitivity is relatively low. Overall, the
methods described can only detect the presence of mutations
and cannot determine the mutated nucleotide sequences.
Using High-Throughput Sequencing to Determine
Targeted Mutations
High-throughput sequencing (deep sequencing) of the whole
genome or single/multiple PCR amplicons is suitable for detect-
ing rare (low frequency) mutations and complicated chimeric mu-
tations, especially for identifying possible off-target mutations
over the whole genome (Fauser et al., 2014; Feng et al., 2014).
However, this strategy is costly and time-consuming.
Using Sanger Sequencing to Determine Targeted
Mutations
A PCR amplicon containing targeted site(s) can be cloned and
examined by Sanger sequencing of multiple clones (about five
clones for uniform mutations [all cells of a plant have the same
mutation] or more than 10 clones for chimeric [multiple] mutations
in somatic cells of a plant). If a restriction site is present at the
cleavage site, the mutant DNA can be enriched using the restric-
tion enzyme digestion method (Shan et al., 2013). This strategy is
useful for determining either simple mutations or complicated
chimeric (mosaic) mutations, but is tedious and expensive.
In some plant species, such as rice, CRISPR/Cas9-based
genome editing is highly efficient and can produce high propor-
tions of uniform mutations (including biallelic, homozygous, or
heterozygous mutations) in the first transgenic generation (T0)
(Zhang et al., 2014; Zhou et al., 2014a; Ma et al., 2015b). This
high-efficiency editing makes the pre-assays described above
for detecting the presence of mutations unnecessary; the
PCR amplicons can be directly sequenced to determine the
actual nucleotide variations in transgenic (T0 and/or T1) plants.
However, without cloning into a plasmid, direct sequencing of
PCR amplicons containing biallelic and heterozygous mutations
results in superimposed sequencing chromatograms. To resolve
this problem, a method called degenerate sequence decoding
(DSD) (Ma et al., 2015a), and its web-based tool DSDecode
(http://dsdecode.scgene.com/) (Liu et al., 2015), have been
developed, which can rapidly determine the mutated allelic
sequences from the sequencing files (ab1 format) with
superimposed chromatograms, thus avoiding the tedious and
expensive cloning and multiclone sequencing. This tool greatly
facilitates the analysis of targeted sites.
EFFICIENCY AND FEATURES OF CRISPR/
CAS9-INDUCED MUTATIONS IN PLANTS
The efficiency and features of CRISPR/Cas9 genome editing in
plants depend on many factors, including the vector system
(the Cas9 gene and the used promoters driving Cas9 and
sgRNAs), target sites, targeted plants, and transformation
methods.
CRISPR/Cas9 Platforms for Plant Genome Editing
Figure 3. Various Types of Targeted Mutations by Different
Mechanisms.
(A) After creating DSBs in the target sites of both homologous chromo-
somes by the Cas9/sgRNA complex, the repair by NHEJ may produce
heterozygous mutation, biallelic mutations (two distinct mutations), or
homozygous mutation (two independent identical mutations).
(B) An NHEJ–HDR pathway for producing a homozygous mutation. A DSB
occurs at one of the two chromosomal sites and a mutation is produced.
Then a DSB occurs at another chromosomal site followed by an HDR
process with the first mutated chromosome as the template.
Magenta dashes indicate the cutting site of Cas9 protein; ‘‘+’’ and ‘‘*’’ both
indicate small indels mutation, but they are used for representing different
indels; Cross lines indicate the process of ‘‘homologous-directed repair,
HDR’’.
Factors Affecting the Efficiency and Inheritance of the
Targeted Mutations
Although CRISPR/Cas9-mediated genome editing has been
extensively applied in various plants, the editing efficiencies
vary dramatically (Bortesi and Fischer, 2015). The codon
optimization of Cas9, the promoter for Cas9 expression, and
the position effect of the T-DNA insertions may directly affect
Cas9 expression levels and the targeting efficiencies (Yan et al.,
2015; Ma et al., 2015b; Wang et al., 2015b; Mao et al., 2016).
The stable heredity of CRISPR/Cas9-induced mutations has
been confirmed by a number of reports (Feng et al., 2014;
Zhang et al., 2014; Zhou et al., 2014a; Ma et al., 2015b). In
general, uniform mutations are more reliably transmitted into
the progeny than into the chimeric mutations.
Previous reports on transient expression assays indicate that
the sgRNA level can limit the editing efficiency (Jinek et al.,
2013; Li et al., 2013). However, for Agrobacterium-mediated
stable transformation approaches, varied sgRNA levels driven
by different U3 and U6 promoters in rice produced similar
editing efficiencies (Ma et al., 2015b), suggesting that in most
cases the sgRNA level is sufficient for the efficient function of
the Cas9/sgRNA complex in rice.
In rice, high proportions (up to 86% on average) of targeted
mutations can be obtained in the T0 transgenic plants; about
Molecular Plant
half of these are biallelic (two distinct allelic mutations) and the
remainder are homozygous or heterozygous (Figure 3A), with
rare chimeric mutations (Zhang et al., 2014; Ma et al., 2015b). It
is noteworthy that the homozygous mutations occur in a higher
proportion than expected in T0 rice plants. It is proposed that
the homozygous mutations may result from two mechanisms.
First, NHEJ may induce two independent identical mutations at
both homologous chromosomal sites (Figure 3A), especially for
the high-frequency ‘‘A’’ and ‘‘T’’ base insertions; second, the
first mutated allele is created by NHEJ, and then may serve as
a template for HDR of the other homologous site to produce
the same mutation (Ma et al., 2015b) (Figure 3B).
In contrast to rice and other plants, Arabidopsis T1 plants derived
from constructs using constitutive promoter-driven Cas9 and
Agrobacterium-mediated floral dip transformation show much
lower efficiency of mutations and usually produce mosaic plants
(Mao et al., 2013; Feng et al., 2014; Jiang et al., 2014; Yan et al.,
2015; Ma et al., 2015b). This lower editing efficiency in
Arabidopsis may be due to the relatively low expression levels
of Cas9 driven by the constitutive promoters in the embryo sac,
which is the primary target of Agrobacterium infection in the
floral dip method (Desfeux et al., 2000). To improve genome
editing in Arabidopsis, several studies used tissue-specific
promoters to drive Cas9, including the anther-specific DD45
promoter (Mao et al., 2016), the egg cell-specific EC1.2 promoter
(Wang et al., 2015b), the INCURVATA2 promoter (Hyun et al.,
2015), and the meristem-specific YAO promoter (Yan et al.,
2015). These modifications improved the mutagenesis in T1
or T2 plants in Arabidopsis. In addition, the terminator of the
Cas9 expression cassette was proposed to affect the editing
efficiency (Wang et al., 2015b).
Effects of Target-Site Selection on Editing Efficiency
The U3 and U6 promoters used for sgRNA expression have defi-
nite transcription initiation sites (A nucleotide for U3 promoters
and G nucleotide for U6 promoters). Early studies restricted the
first nucleotide to A or G in the selected targets, as the regular
forms AN19NGG (for the U3 promoters) and GN19NGG (for the
U6 promoters) (Jinek et al., 2013; Mali et al., 2013). However,
recent studies show that sgRNAs with one or a few extra
nucleotides (irregular sgRNAs) in the 50 termini also can mediate
genome editing (Ran et al., 2013; Xie and Yang, 2013; Ma et al.,
2015b), and a comparison of large number of the irregular
sgRNAs with the regular sgRNAs showed similar editing
efficiency between them (Ma et al., 2015b). Thus, targets with
an initial T or C nucleotide also can be used for genome editing,
broadening the possibilities for target selection (Ma et al., 2015b).
The target sequence composition (such as GC content) may also
affect the CRISPR/Cas9-induced editing efficiency (Zhang et al.,
2014; Ma et al., 2015b). In rice, the target sequences with
higher GC contents (50–70%) produce relatively higher editing
efficiencies (Ma et al., 2015b). Furthermore, the formation of a
stem-loop structure of the target sequence with the sgRNA scaf-
fold sequence may affect the binding to the genomic target
strand, and thus may result in a lower editing efficiency (Ma
et al., 2015b). Therefore, genomic sites that pair with the
sgRNA scaffold sequence for more than six continuous
nucleotides should not be used as target sites (Ma et al., 2015b).
Molecular Plant 9, 961–974, July 2016 ª The Author 2016. 969
Molecular Plant
Some Considerations on Off-Target Mutations
Eukaryotic organisms have relatively large genomes with dupli-
cated homologous sequences. A crucial concern in CRISPR/
Cas9-based eukaryotic genome editing is the effect of off-target
mutations, especially for medical research and clinical application.
It was shown that target sequences with very high GC contents
may produce a higher off-targeting effect (Fu et al., 2013). The
nucleotides in PAM and 10-nt guide sequence immediately up-
stream of PAM highly determine the targeting specificity, while
target-like homozygous sequences with variation of a few nucleo-
tides in the PAM-distal region (1–10 nt) may easily cause off-target
mutations (Jinek et al., 2012; Cong et al., 2013; Hsu et al., 2013;
Jiang et al., 2015). Therefore, the risk of off-targeting can be
minimized or avoided by selection of highly specific target se-
quences by genome searching (nucleotide BLAST), or using the
web-based tools for CRISPR/Cas9 target selection (Lei et al.,
2014; Xie et al., 2014). In addition, efforts have been made to
optimize the targeting specificity of the CRISPR/Cas9 system,
for example by modification of the Cas9 gene (Kleinstiver et al.,
2016; Slaymaker et al., 2016), use of a pair of Cas9-derived
nick-nuclease/sgRNAs (Ran et al., 2013), or change of some
nucleotides in the sgRNA sequence (Dang et al., 2015).
For applications of genome editing in gene functional studies of
plants, if off-target mutation(s) occur it may be necessary to
find out whether the off-target mutations cause phenotypic vari-
ation(s) that interfere with analysis of the targeted mutation on the
phenotype. The correlation between the mutant phenotype(s)
and the targeted/off-targeted mutations can be determined by
crossing the mutants with the parental plants followed by co-
segregation analysis in the progenies. In crop molecular breeding
by targeted gene editing, possible off-target mutations may have
three types of consequence on agronomic traits, namely negative
effect, no effect (neutral), and positive effect, that produce better
phenotype(s) of trait(s). The gene-edited plants (lines) with off-
target mutation(s) of negative effect will be discarded during the
breeding selection process because of the negative phenotypes,
or if necessary by crossing with the parental plants followed by
phenotype/marker-aided selection in the progenies. However, if
the off-target mutations are neutral or positive on the trait(s),
these mutations are substantially equivalent to the targeted mu-
tations as well as natural and induced mutations, and can be re-
tained in the new breeding lines. Therefore, it is not necessary to
exclude all off-target mutations in the breeding programs.
Features of CRISPR/Cas9-Induced Mutations
Several reports show that about half of CRISPR/Cas9-induced
mutations are single-base (mostly A and T) insertions and the
rest are small deletions (1–50 bp), while base substitution and
insertion of two or more bases are very rare (Feng et al., 2014;
Zhang et al., 2014; Ma et al., 2015b). Therefore, if the target
sites are located within coding regions of genes, most of the
editing events cause frame-shift and loss-of-function of the
genes. If two or more sites are targeted in a gene or a chromo-
somal region, a fragmental deletion (up to hundreds of kilobases
in length) can occur between the target sites (Li et al., 2013; Mao
et al., 2013; Xie and Yang, 2013; Zhou et al., 2014a; Ma et al.,
2015b; Zhao et al., 2016). This feature is useful for deletion of
whole genes and certain chromosomal regions containing
multiple genes.
970 Molecular Plant 9, 961–974, July 2016 ª The Author 2016.
CRISPR/Cas9 Platforms for Plant Genome Editing
APPLICATION OF CRISPR/CAS9 FOR
EDITING IMPORTANT TRAITS IN PLANTS
The CRISPR/Cas9-based genome editing system has many ap-
plications for functional studies of plant genes. In addition,
CRISPR/Cas9 also provides a robust tool for genetic improve-
ment of important traits in crops, such as yield, architecture,
nutrient usage, disease resistance, and adaption to stresses. In
particular, the traits controlled by negative regulatory genes can
be improved simply by knockout or weakening of the genes.
For example, simultaneous knockout of the three TaMLO homo-
logs in common wheat produced resistance to powdery mildew
(Wang et al., 2014). Targeted mutation of the ERF transcription
factor gene OsERF922 (Liu et al., 2012) in rice enhanced
resistance to rice blast fungal pathogen (Wang et al., 2016).
Also, knockout of TMS5 (Zhou et al., 2014b) in rice cultivars
could rapidly breed temperature-sensitive lines for hybrid rice
production (unpublished results of C.X. Zhuang). Table 2
summarizes the reported applications in a number of plant
species.
CRISPR/Cas9 has also been applied to woody plants; the
ability to obtain phenotypic mutants in the T0 generation is
especially important for woody plants that have very long
reproductive cycles (Fan et al., 2015; Tsai and Xue, 2015). In
addition, the CRISPR/Cas9 system was further explored as
an antivirus tool to repress DNA virus infection in the host
plants by specifically cleaving the virus DNAs (Baltes et al.,
2015; Chaparro-Garcia et al., 2015; Ji et al., 2015; Ali et al.,
2015b).
Targeted gene knock-in and replacement via HDR has many
highly desirable applications in biological research and genetic
improvements in crop breeding, because this technology can
precisely edit target genes or insert genes or fragments into
target sites. Although gene knockout using CRISPR/Cas9 has
become feasible with high efficiency in many plants, so far
only a few cases of CRISPR/Cas9-based gene knock-in/
replacement in plants have been reported. For example,
knock-in/replacement editing using CRISPR/Cas9 and particle
bombardment or Agrobacterium infection were successfully ob-
tained in Arabidopsis, maize, rice, and soybean (Table 2) (Schiml
et al., 2014; Li et al., 2015; Endo et al., 2016; Sun et al., 2016;
Zhao et al., 2016). To boost HDR-mediated precise editing, a
geminivirus system was used to amplify the template donor
sequence (Cermak et al., 2015). Also, knock-in/replacement
editing can be further promoted by suppression of DNA ligase
IV, which participates in the NHEJ pathway (Endo et al., 2016;
Nishizawa-Yokoi et al., 2016). However, due to the relatively
low efficiency of the HDR pathway in plants and inefficient
delivery of homologous donor sequences into transfected
plant cells (Puchta and Fauser, 2014), at present knock-in/
replacement editing in plants remains difficult and of low
efficiency. The few successful cases of knock-in/replacement
editing in plants mainly involve herbicide- or antibiotic-
resistance genes, because these edited genes can give the cells
a growth advantage under selection pressure (Li et al., 2015;
Svitashev et al., 2015). Nevertheless, these advancements
shed light on future improvements and extensive applications
of this technology for plant research and molecular breeding in
crops.
CRISPR/Cas9 Platforms for Plant Genome Editing
CONCLUDING REMARKS AND
PERSPECTIVES
CRISPR/Cas9 genome editing technology will have a revolution-
ary effect on plant research and crop breeding (Belhaj et al., 2015;
Ma and Liu, 2016; Weeks et al., 2015). Compared with medical
and clinical research, genome editing in plants does not involve
ethical issues, and thus is more suitable for applied research. In
principle, CRISPR/Cas9-induced mutations are substantially
equivalent to those produced by random mutagenesis, but
have much higher specificity and efficiency. In addition, the trans-
genic Cas9 gene and the antibiotic-resistant marker genes can be
simply removed by segregation in the progenies, producing
transgene-free gene-edited plant lines (Zhou et al., 2014a;
Lawrenson et al., 2015).
Although a number of CRISPR/Cas9 vector platforms have been
established and tested for their effectiveness in genome editing,
the potential of the CRISPR/Cas9 system remains far from fully
explored. Current modifications of CRISPR/Cas9 aim to increase
its efficiency, specificity, and range of accessible targets. Some
studies have shown that the PAM-determining domain of Cas9
can be modified to anchor other PAM motifs (Kleinstiver et al.,
2015a, 2015b; Hu et al., 2016). In addition, modification of the
sgRNA, such as extending the duplex length and mutating the
fourth thymine of the polythymine sequence to cytosine or
guanine, can also improve the editing efficiency (Dang et al.,
2015). However, further establishment and improvement of
knock-in/replacement-based precise editing to any gene in
target plants remains a major challenge.
In addition, several other orthologs of SpCas9, such as StCas9
(Cong et al., 2013) and a smaller SaCas9 (Ran et al., 2015),
have been identified and confirmed to be functional in plants
(Steinert et al., 2015). The newly developed Cpf1, which was
also adapted from the Class II CRISPR system, can create
DSBs with sticky ends (Zetsche et al., 2015). Moreover, Cas9
has been modified with a gene activation or repression domain
and used for regulation of gene expression (Gilbert et al., 2013).
Expanding this idea, a modified CRISPR/Cas9 toolbox for plant
gene activation and repression has been developed (Lowder
et al., 2015). New tools derived from the CRISPR/Cas9 or
similar systems may also be explored for novel applications
such as RNA cleavage (O’Connell et al., 2014), epigenomic
regulation (Hilton et al., 2015; Kearns et al., 2015), and
chromatin imaging (Chen et al., 2013) in plants in the future.
Given its simplicity, flexibility, versatility, and efficiency, the
CRISPR/Cas9 genome editing system presumably will
exceed the capabilities of previous genome manipulation technol-
ogies. As one of the top ten breakthroughs selected by Science
magazine (http://news.sciencemag.org/scientific-community/2015/
12/and-science-s-breakthrough-year), CRISPR/Cas9 and other
emerging genome editing systems will revolutionize biology
research and crop molecular breeding in the coming years.
FUNDING
This work was supported by grants from Guangdong Province Public In-
terest Research and Capacity Building Special Fund (2015B020201002)
and the Ministry of Agriculture of the People’s Republic of China
(2014ZX08010-001, 2014ZX08009002).
Molecular Plant
ACKNOWLEDGMENTS
No conflict of interest declared.
Received: March 23, 2016
Revised: April 14, 2016
Accepted: April 15, 2016
Published: April 19, 2016
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