Review Article

https://doi.org/10.1038/s41477-019-0461-5

The emerging and uncultivated potential of

CRISPR technology in plant science
Yingxiao Zhang1, Aimee A. Malzahn1, Simon Sretenovic1 and Yiping Qi   1,2*

The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolution-
ized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea,
and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process,
various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing,
epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appre-
ciated and solutions explored. This Review intends to provide an informative summary of the latest developments and break-
throughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not
only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to
improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural,
environmental and ecological challenges.

G
enerating targeted genetic changes in living cells and organ-
isms has historically been a great challenge in many species.
The basic strategy to introduce genetic changes is to gen-
erate double-strand breaks (DSBs) by a sequence-specific nucle-
ase (SSN) at the targeted genomic site. Repair of DSBs in somatic
cells of higher eukaryotes is predominantly done through the non-
homologous end joining (NHEJ) pathway, which results in inser-
tions/deletions (indels). Homology-directed repair (HDR), as a
minor pathway, helps achieve precise genomic changes. Early SSNs,
including zinc finger nucleases (ZFNs)1 and transcription activator-
like effector nucleases (TALENs)2, rely on protein–DNA interac-
tions to determine specificity, which are difficult to engineer and
multiplex. In 2012, the clustered regularly interspaced short palin-
dromic repeat (CRISPR)–CRISPR-associated (Cas) system was first
used for programmable RNA-guided genome editing in vitro3. The
following year, genome editing using CRISPR was successfully dem-
onstrated in a mammalian system4,5. CRISPR, as a versatile, simple
and inexpensive system for genetic manipulation, has since domi-
nated the genome editing field.
Since the first demonstration of CRISPR in plant genome edit-
ing in 2013 (refs. 6–8) there has been much progress in basic plant
science and crop improvement9,10. Numerous molecular tools and
platforms have been developed to achieve plant genetic engineering,
including targeted mutagenesis11–13, base editing14, precise editing by
HDR15 and transcriptional regulation16. In this Review, we describe
the state-of-the-art development of CRISPR technologies that have
been applied in plants or remain to be fully explored, which are
partly illustrated in Fig. 1. We cover available CRISPR nucleases and
their variants, expression systems for multiplexing, diverse ways
to achieve precise genome editing, epigenome editing and tran-
scriptome regulation. We address specific considerations in plant
genome editing, such as temperature sensitivity, transgene-free
editing, polyploid genome editing and germline editing through
floral dip. Recent breakthroughs that combine CRISPR with other
cutting-edge technologies in plant breeding are also presented. We
envision some new frontiers where CRISPR-based plant genome
engineering will likely head. We hope this Review provides a com-
prehensive guide on the current status and some future directions of
CRISPR technology in plants.
The diverse and expanding CRISPR toolbox
CRISPR arrays exist in bacteria and archaea DNA sequences,
consisting of short repeats separated by unique spacer sequences.
Small clusters of genes encoding Cas proteins can be found around
CRISPR arrays. Cas proteins are used to classify diverse CRISPR
systems based on their phylogenetic, structural and functional
characteristics. During CRISPR antiviral defence, if the pre-
CRISPR RNA (pre-crRNA) processing and interference stages are
accomplished by one single multifunctional protein, the CRISPR
system is grouped as class 2; otherwise, it is grouped as a class 1
system17. Each class can be divided into multiple types according
to their signature proteins: type I, III and IV belong to class 1, with
Cas3, Cas10 and Csf1 as their respective signature proteins17; while
type II (Cas9), type V (Cas12a–e (Cas12d and Cas12e are also
known as CasY and CasX, respectively), Cas12g–i and Cas14a–c)
and type VI (Cas13a–d) belong to class 2 (refs. 18–23). Each type of
CRISPR system can be further grouped into subtypes based on
operon organization and Cas proteins at the CRISPR loci. Although
the classification of CRISPR systems is constantly under develop-
ment, the current established classification method will facilitate
our understanding of CRISPR systems and the discovery of new
Cas proteins.
CRISPR–SpCas9 genome editing system. Cas9 is a class 2, type
II CRISPR system and has been adapted for genome editing in the
vast majority of organisms. Targeted genome cleavage requires Cas9
to assemble with a single-guide RNA (sgRNA; a fusion of crRNA
and trans-activating crRNA (tracrRNA)), then recognize and bind
to desired DNA sequences followed by a protospacer adjacent motif
(PAM). Streptococcus pyogenes Cas9 (SpCas9) recognizes a very sim-
ple PAM (NGG), making it the most commonly used CRISPR–Cas9
system. SpCas9 has been codon-optimized for different species,

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA. 2Institute for Bioscience and Biotechnology
1

Research, University of Maryland, Rockville, MD, USA. *e-mail: yiping@umd.edu

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NATURe PlAnTS Review Article
Plasmid dsDNA RNA RNP

Attachment Viral vector

Nanoparticle
PEG
Particle
Agrobacterium bombardment O H
H O
n

RNA editing

ºC Temperature sensitivity
Organelle genome editing
90
High activity 80
70
RNA base editing 60
50
40
Low activity 30
20

C
as
C
10

as
Mitochondria

12
C

12

b
as
Plastid

a
9
Cyto
plas
m
Introduce Nuc
leus
stop codon
Gene knockout
D
Regulatory elements Promoter 5′ UTR Exon1 Intron Exon2 3′ UTR Terminator

Precise genome editing (base editing)

D D D D D
Regulatory elements Promoter 5′ UTR Exon1 Intron Exon2 3′ UTR Terminator

Precise genome editing (HDR)

Regulatory elements Promoter 5′ UTR Exon1 Intron Exon2 3′ UTR Terminator

LHA RHA
Transcriptional regulation
D Edit splicing sites
Regulatory elements Promoter 5′ UTR Exon1 Intron Exon2 3′ UTR Terminator

D R

Cas Cas Dead FnCas9 Cas13 gRNA Deaminase Regulator ADAR

nickase Cas

Fig. 1 | Applications of CRISPR technology in plant cells. Outside the cell, CRISPR reagents can be delivered as plasmids, dsDNA, RNA and RNPs
through PEG-mediated transformation, particle bombardment or nanoparticles. Plasmids can also be delivered by Agrobacterium and viral vectors. In the
cytoplasm, CRISPR can cleave and edit RNA molecules through RNA-targeting Cas proteins, such as FnCas9 and Cas13, and the ADAR-derived RNA base
editor, respectively. CRISPR may also be introduced into organelles to edit organelle genomes. Commonly used Cas systems (Cas9, Cas12a and Cas12b)
are temperature sensitive, requiring higher temperatures for optimal activities. In the nucleus, CRISPR technology is applied to achieve gene knockout,
precise genome editing through base editing, HDR and transcriptional regulation. To knock out genes, CRISPR–Cas nucleases can be used to target coding
regions or regulatory elements. Base editors can be used to target coding regions (for example, to introduce stop codons) or regulatory elements. When
the DNA repair donor with the left homology arm (LHA) and the right homology arm (RHA) are introduced along CRISPR–Cas, precise genome editing
can be achieved through HDR. To regulate transcription, CRISPR–Cas nucleases and base editors can be used to edit regulatory elements or splicing sites.
Transcriptional regulation can also be achieved by using dead Cas proteins to recruit regulators to the promoter region, including activators, repressors,
DNA methyltransferase, demethylase and so on.

including human (Homo sapiens; hCas9)5, plant (pcoCas9 and
Cas9p)6,24, Arabidopsis thaliana (AteCas9)25, maize (Zea mays;
zCas9)26,27 and soybean (Glycine max; GmCas9)28, to improve the
expression level in vivo. By introducing the point mutations D10A
in the RuvCI domain or H840A in the HNH domain, a Cas9 nickase
(nCas9) is generated that only cleaves the targeting or non-targeting
strand, respectively4,29,30. When both mutations are introduced, the
nuclease activity is abolished, resulting in a catalytically inactive or
dead Cas9 (dCas9)31. Cas9 nuclease, nCas9 and dCas9 can all be
applied to develop many versatile genome engineering tools.

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Review Article
Expanding target ranges with Cas9 variants and orthologues.
The PAM requirement of SpCas9 limits its targeting scope in a
genome. The PAM-interacting (PI) domain can be engineered
through rationale design and directed evolution to obtain mutated
versions with altered PAM requirements (Table 1). The SpCas9
VQR, EQR and VRER variants can robustly recognize NGA,
NGAG and NGCG PAMs, respectively32. Similarly, the QQR1
variant was engineered with highly specific binding to the NAAG
PAM33. Using phage-assisted continuous evolution, xCas9 has been
developed, recognizing NG, GAA and GAT PAM sites34. In addi-
tion, a rationally engineered SpCas9-NG recognizes relaxed NG
PAMs and outperforms xCas9 at sites with NGH PAMs35. Many of
these SpCas9 variants have been demonstrated in plants, such as
the VQR variant36,37 and the VRER variant36 in rice (Oryza sativa),
as well as xCas9 and SpCas9-NG in rice and Arabidopsis38–46. Both
xCas9 and SpCas9-NG have shown lower editing activity than wild-
type SpCas9 at NGG PAMs while exhibiting higher activity at non-
canonical NG PAMs39,40,46. Specifically, SpCas9-NG is superior to
xCas9 at most target sites with NG PAMs, as well as AT-rich PAM
sites such as GAT, GAA and CAA39,40. Although the performance of
some Cas9 variants may be suboptimal in plants, further optimiza-
tion could help restore editing activity in a plant cellular environ-
ment to achieve efficient genome editing.
Alternative PAM recognition can also be achieved by harnessing
Cas9 orthologues from other bacterial and archaeal species (Table 1).
Staphylococcus aureus Cas9 (SaCas9) recognizes an NNGRRT
PAM and is smaller in size compared to SpCas9, making it pre-
ferred for virus-based delivery47. SaCas9 nickase48, dead SaCas9
(ref. 47) and a KKH variant49 with a loosened PAM requirement
have been developed to expand the application of this system.
SaCas9 has been demonstrated in multiple plant species, including
Nicotiana benthamiana50, rice50, Arabidopsis51 and citrus52. Other
Cas9 orthologues that have been used for genome editing in mam-
malian systems include Streptococcus thermophilus Cas9 (St1Cas9)
and St3Cas9 (ref. 53); Neisseria meningitidis Cas9 (NmCas9)54;
Francisella novicida Cas9 (FnCas9) and its RHA variant55,
Treponema denticola Cas9 (TdCas9)56; Campylobacter jejuni Cas9
(CjCas9)57 and Streptococcus canis Cas9 (ScCas9)58, of which
only St1Cas9 has been demonstrated to edit plant genomes
(Arabidopsis)51. Brevibacillus laterosporus Cas9 (BlatCas9) has also
been characterized and is used for maize genome editing59. Notably,
Cas9s from class 2 type II-A and II-C have shown RNA-guided sin-
gle-stranded RNA cleavage activity60. Moreover, by grafting the PI
domain of a Streptococcus macacae Cas9 (SmacCas9) to SpCas9,
iSpy-macCas9 has been developed to recognize NAA PAM61. In
general, the availability of Cas9 orthologues increases the accessible
target sites for genome editing. Cas9 orthologues can be used for
orthogonal genome engineering, benefiting from their distinctive
sgRNA structures.
Cas12a is a distinct CRISPR system. Cas12a (formerly known as
Cpf1, CRISPR from Prevotella and Francisella 1), a class 2 type V
endonuclease, is a genome editing system that is distinct from Cas9
(ref. 62). First, the PAM requirement allows Cas12a to target T-rich
regions62. Second, Cas12a only requires a short crRNA (~42 nt),
making Cas12a crRNA easy to synthesize, multiplex and engineer62.
In addition to DNA nuclease activity, Cas12a also possesses RNase
activity, which is able to process a CRISPR array for multiplexed
genome editing62,63. Moreover, Cas12a generates a DSB with stag-
gered ends distal from the PAM site, which allows continuous
cleavage of DNA and may promote NHEJ-based gene insertion62.
Cas12a is also considered more specific than wild-type SpCas9
in several biological systems64–67. Three Cas12a nucleases from
Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium
ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) have been
demonstrated in rice66–72, Arabidopsis73, tobacco (N. benthamiana
780
NATURe PlAnTS
and Nicotiana tabacum)68,73,74, tomato (Solanum lycopersicum)73,
soybean74, cotton (Gossypium hirsutum)75 and citrus76.
Similar to Cas9, Cas12a also contains a recognition (REC) lobe
and a nuclease (NUC) lobe63. However, the introduction of muta-
tions at the catalytic residues in the RuvC domain of Cas12a abol-
ishes the cleavage of both DNA strands at target sites62,63. Therefore,
there is no Cas12a nickase currently available. Multiple versions
of catalytically dead Cas12a (dCas12a) have been engineered and
repurposed for different applications, including dAsCas12a, dLb-
Cas12a and DNase-dead Cas12a (namely ddCas12a)62,63,66,77. To
broaden the target ranges of Cas12a, mutations have been intro-
duced to the wedge and PI domains to generate RR and RVR vari-
ants78,79 (Table 1). Similar mutations have been further introduced
into rice-codon-optimized LbCas12a and FnCas12a for genome
editing in plants; only LbCas12a variants successfully edit sites with
altered PAMs in plants67,80. Recently, an enhanced AsCas12a variant
(enAsCas12a) has been engineered to recognize a broader range of
PAM sites and also exhibits enhanced activity at low temperatures81.
To further expand the utility of Cas12a in genome editing, ortho-
logues from diverse bacteria species have been surveyed62,82 (Table 1).
Cas12a orthologues with editing activity at loosened or shorter
PAM sites include Moraxella bovoculi 237 Cas12a (MbCas12a),
M. bovoculi AAX08_00205 Cas12a (Mb2Cas12a), M. bovoculi
AAX11_00205 Cas12a (Mb3Cas12a), Thiomicrospira sp. XS5 Cas12a
(TsCas12a), Btyrivibrio sp. NC3005 Cas12a (BsCas12a), Helcococcus
kunzii ATCC 51366 Cas12a (HkCas12a), Lachnospira pectinoschiza
strain 2789STDY5834886 Cas12a (LpCas12a), Pseudobutyrivibrio
ruminis CF1b Cas12a (PrCas12a) and Pseudobutyrivibrio xylaniv-
orans strain DSM 10317 Cas12a (PxCas12a)62,79,82,83, which could
potentially be useful for plant genome editing.
Emerging DNA-targeting CRISPR nucleases. Cas12b, formerly
known as C2c1, is a class 2 type V-B endonuclease. Cas12b recog-
nizes target sequences with a distal 5′-T-rich PAM sequence and
generates staggered DSBs with 7-nt 5′-overhangs21,84. Like Cas9,
Cas12b requires both crRNA and tracrRNA for target recogni-
tion, which can be engineered as a sgRNA21. Like Cas12a, Cas12b
contains a REC lobe and a NUC lobe without a HNH domain85.
Two Cas12b nucleases, Alicyclobacillus acidoterrestris Cas12b
(AacCas12b) and Bacillus thermoamylovorans Cas12b (BthCas12b),
have initially shown editing activity in vitro with a preference for
higher temperatures (48–50 ℃)21, which is not ideal for mamma-
lian and plant genome editing. By exploring diverse Cas12b ortho-
logues, Alicyclobacillus acidiphilus Cas12b (AaCas12b) has been
discovered and shows efficient editing activity at a broader range
of temperatures86. Later, Bacillus hisashii Cas12b (BhCas12b) was
screened from multiple Cas12b orthologues and further engineered
to increase activity at lower temperatures87. The Cas12b orthologues
that possess nuclease activity at lower temperatures may be applied
for plant genome editing.
CasX (also known as Cas12e), which was discovered from pre-
viously uncultivated environmental microbial communities, is a
group of dual-RNA-guided Cas proteins targeting double-stranded
DNA (dsDNA) with a 5′-TTCN PAM18,20. CasX requires a 20 nt
guide RNA (gRNA) and a tracrRNA, and its cleavage generates a
staggered-end break with an overhang of approximately 10 nt20.
Deltaproteobacteria CasX (DpbCasX) and Planctomycetes CasX
(PlmCasX) have been shown to edit the genomes of Escherichia
coli and human cells20. Mutations can be introduced to the RuvC
domain to generate deactivated CasX20. Additionally, another
CRISPR system, CasY (also known as Cas12d), has also been identi-
fied but is less well characterized. CasY may recognize a 5′-TA PAM
and cleave dsDNA18.
Researchers further exploited class 1 CRISPR systems for genome
editing. A type I-E CRISPR system from Thermobifida fusca has
been shown to achieve 13–60% editing efficiency in mammalian
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NATURe PlAnTS Review Article
Table 1 | Variants and orthologues of Cas9 and Cas12a
Cas Size (amino PAM Mutations Plants Features
acids)
SpCas9 (refs. 3–5) 1,368–1,424 NGG – Many plant species –
SpCas9 VQR 32
1,372 NGA D1135V/R1335Q/T1337R Rice 36,37
Altered PAM
SpCas9 EQR32 1,372 NGAG D1135E/R1335Q/T1337R – Altered PAM
SpCas9 VRER32 1,372 NGCG D1135V/G1218R/R1335E/T1337R Rice5 Altered PAM
SpCas9 D1135E4 1,372 NAG and NGA D1135E – Altered PAM
SpCas9 QQR1 (ref. 33) 1,372 NAAG G1218R/N1286Q/I1331F/D1332K/ – Altered PAM
R1333Q/R1335Q/T1337R
SpCas9-NG35 1,372 NG R1335V/L1111R/D1135V/G1218R/ Rice39,40,42,43 and Arabidopsis42 Altered PAM
E1219F/A1322R/T1337R
iSpy-macCas9 (ref. 61) 1,359 NAA R221K/N394K – Altered PAM
SpCas9-HF1 (ref. 253) 1,368 NGG N497A/R661A/Q695A/Q926A Rice259,260 and Arabidopsis261 Enhanced specificity
SpCas9 (K855A)254 1,424 NGG K855A – Enhanced specificity
eSpCas9 (1.0)254 1,424 NGG K810A/K1003A/R1060A Rice259 and Arabidopsis261 Enhanced specificity
eSpCas9 (1.1)254 1,424 NGG K848A/K1003A/R1060A Rice259 and Arabidopsis261 Enhanced specificity
HypaCas9 (ref. 255) 1,368 NGG N692A/M694A/Q695A/H698A Rice260 Enhanced specificity
eHF1-Cas9 (ref. 260
) 1,368 NGG N497A/R661A/Q695A/K848A/ Rice 260
Enhanced specificity
Q926A/K1003A/R1060A
eHypa-Cas9 (ref. 260) 1,368 NGG N692A/M694A/Q695A/H698A/ Rice260 Enhanced specificity
K848A/K1003A/R1060A
EvoCas9 (ref. 256) 1,368 NGG M495V/Y515N/K526E/R661Q – Enhanced specificity
Sniper-Cas9 (ref. 257) 1,372 NGG F539S/M763I/K890N – Enhanced specificity
HiFi Cas9 (ref. 258) 1,368 NGG R691A – Enhanced specificity
xCas9 3.7 (ref. 34) 1,368 NG, GAA A262T/R324L/S409I/E480K/ Rice38–40 Enhanced specificity
and GAT E543D/M694I/E1219V and altered PAM
SaCas9 (refs. 47,297) 1,053 NNGRRT – N. benthamiana50,298, –
Arabidopsis51, rice50 and citrus52
SaCas9 KKH49 1,053 NNNRRT E782K/N968K/R1015H – –
St1Cas9 (ref. 53) 1,122 NNAGAAW – Arabidopsis51 –
St3Cas9 (ref. 53) 1,393 NGGNG – – –
NmCas9 (ref. 54) 1,109 NNNNGATT – – –
FnCas9 (ref. 55) 1,629 NGG – – –
FnCas9 RHA55 1,632 YG E1369R/E1449H/R1556A – –
TdCas9 (ref. ) 56
1,423 NAAAAN – – –
CjCas9 (ref. 57) 984 NNNNACAC or – – –
NNNNRYAC
ScCas9 (ref. 58) 1,379 NNG – – –
SmacCas9 (ref. 61) 1,338 NAA – – –
BlatCas9 (ref. 59) 1,092 NNNNCND – Maize59 –
AsCas12a 62
1,307 TTTV – Rice66, N. benthamiana and –
tomato73, soybean and wild
tobacco74
AsCas12a RR78 1,307 TYCV and S542R/K607R – Altered PAM
CCCC
AsCas12a RVR78 1,307 TATV S542R/K548V/N552R – Altered PAM
enAsCas12a81 1,307 VTTV, TTTT, E174R/S542R/K548R – Altered PAM and
TTCN and TATV enhanced activity at
low temperature
LbCas12a62 1,228 TTTV – Rice66–72, Arabidopsis73,211, –
N. benthamiana and tomato73,
soybean and wild tobacco74,
cotton75, citrus76 and maize27
LbCas12a RR78 1,228 TYCV and G532R/K595R Rice67,80 Altered PAM
CCCC
Continued

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Table 1 | Variants and orthologues of Cas9 and Cas12a (continued)
Cas Size (amino PAM Mutations
acids)
LbCas12a RVR78 1,228 TATV G532R/K538V/Y542R
FnCas12a62,79
1,300 TTV, TTTV and – Rice67,68,70,72 and tobacco68
KYTV
FnCas12a RR79 1,300 TYCV and N607R/K671R
TCTV
FnCas12a RVR79 1,300 TWTV N607R/K613V/N617R
MbCas12a 62,79
1,373 TTV and TTTV – –
MbCas12a RR79 1,373 TYCV and N576R/K637R
TCTV
MbCas12a RVR79 1,373 TWTV N576R/K582V/N586R
cells88. This system requires a multi-subunit cascade (CRISPR-
associated complex for antiviral defence) and a crRNA to recognize
target sequences with a 5′-PAM; the helicase-nuclease enzyme Cas3
is then recruited to degrade DNA. Cas3 generates a range of large
deletions up to 100 kb, which may be potentially useful for large-
scale genome manipulation and deletion screens.
Cas14 is a family of miniature nucleases (400–700 amino acids
long) that can cleave targeted single-stranded DNA (ssDNA)
without PAM requirements19. Cas14 can be used for diagnosing
diseases19 and may be also applied for ssDNA virus interference
in plants.
Repurposing CRISPR as a recruiting platform. The programmable
DNA-binding property of CRISPR systems provides a powerful plat-
form to recruit functional domains to desired regions of the genome
through protein fusion or sgRNA–protein interactions (Fig. 2).
Such functional domains can be fused to the C terminus or N ter-
minus of Cas proteins and may include FokI nuclease domains,
transcriptional activators or repressors, epigenetic modifiers, deam-
inases, fluorescent protein tags and so on. To increase the dosage
of the functional domains, the SUperNova tagging system (SunTag)
uses a repeat of GCN4 epitopes fused to dCas9, which are bound by
scFv antibodies fused to effectors89. Protein recruitment can also be
achieved using self-complementing split green fluorescent protein
(GFP)90 (Fig. 2a). In contrast to the protein fusion strategy, func-
tional domains can be recruited through RNA–protein interactions
(Fig. 2b). The versatility of Cas9 sgRNA allows for the modification
of the structural modules, including the upper stem, the first and
the second hairpins and the 3'-end of the sgRNA, without compris-
ing binding efficiency. Several RNA-binding protein–RNA aptamer
systems have been used for CRISPR protein recruitment, includ-
ing MCP–MS2 (refs. 29,91–93), PCP–PP7 (refs. 93,94), Com–Com95, λ
N22 peptide–boxB96, as well as streptavidin and its binding RNA
aptamer S1 (ref. 97), and so on. For the most widely used MCP–
MS2 system, up to 16 MS2 loops have been added to the 3′-end
and hairpins of the sgRNA98. Moreover, an octet array of MS2 is
added to the upper stem of the sgRNA to create the CRISPR–Sirius
system, resulting in more stable sgRNA secondary structures99. By
fusing SunTag epitopes to MCP and using MS2 to recruit activators,
a three-component repurposed technology for enhanced expres-
sion (TREE) system has been developed100. These versatile DNA-
targeting and protein-recruiting CRISPR systems demonstrate that
multiple approaches can be used simultaneously to maximize the
number and types of functional domains, expanding genome engi-
neering outcomes in plants for base editing and transcriptional reg-
ulation, which are discussed later in this Review (in sections titled
“Precise genome editing by base editors” and “Transcription regula-
tion and epigenome editing”).
782
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Plants Features
Rice67,80 Altered PAM
–
Rice67 –
Rice67 –
–
– Altered PAM
– Altered PAM
RNA-targeting CRISPR nucleases. In addition to the aforemen-
tioned Cas systems that target DNA, Cas13 (formerly known as C2c2)
is a class 2 type VI CRISPR system targeting RNA101. Leptotrichia
shahii Cas13a (LshCas13a) recognizes a 22–28 nt target sequence
followed by a protospacer flanking sequence (PFS) of H (H denotes
A, U or C), while no specific PFS requirement for Leptotrichia
wadei Cas13a (LwaCas13a) and Prevotella sp. P5-125 Cas13b
(PspCas13b)102,103 has been discovered. Like Cas12a, the RNase activ-
ity of Cas13a allows it to process crRNA arrays, which can be used
to target multiple RNAs simultaneously101. Interestingly, Cas13a
shows non-specific RNase activity that cleaves collateral RNA fol-
lowing initial binding to its target RNA in vitro and in bacteria101,104.
By harnessing this special feature of Cas13a, the specific high-sensi-
tivity enzymatic reporter unlocking (SHERLOCK) method has been
developed to detect specific RNA and DNA sequences104. A catalyti-
cally deactivated Cas13 or dCas13 can be generated by introducing
an alanine substitution to any of the four HEPN catalytic residues
(R597A, H602A, R1278A or H1283A)101. dCas13 has been used as a
programmable RNA binding protein to achieve in vivo RNA track-
ing102 and base editing103. In plants, gene expression knockdown
using LwaCas13a has been achieved in rice protoplasts102. RNA virus
interference using LshCas13a has been demonstrated in N. ben-
thamiana105 and Arabidopsis106. Potentially more Cas13 orthologues
can be tested in plants to develop RNA targeting tools for transcrip-
tional regulation, RNA base editing, RNA tracking and functional
studies, pathogen detection and disease control.
Versatile and multiplexed CRISPR expression systems
There are generally four strategies for in planta expression of the
two CRISPR components, Cas protein and gRNA, and each strategy
has its advantages and disadvantages (Fig. 3a). The most commonly
used system is the mixed dual promoter system in which the Cas
gene is driven by an RNA polymerase II (Pol II) promoter while
the gRNA is driven by an RNA polymerase III (Pol III) promoter107.
Due to limitations of Pol III promoters, a dual Pol II promoter sys-
tem has been developed, which resulted in a high transcription
level of gRNAs66,108,109. Pol II promoters allow for potential spatial
and temporal control of expression, and usually outperform Pol III
promoters for generating long transcripts with multiplexed gRNAs.
This system can be further simplified to a compact single transcript
unit using only one promoter to drive both the Cas protein and
the gRNA110–113. A similar compact system expresses gRNAs in the
intron region, allowing the gRNAs to be excised during splicing113,114.
Finally, a bidirectional promoter can be used to drive Cas and gRNA
expression in opposite directions, with different 3′-untranslated
regions (UTRs) or terminators to regulate both components inde-
pendently. In the future, these four expression strategies may be fur-
ther evaluated and applied for tailored applications in plants.
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NATURe PlAnTS Review Article
a Effector protein recruitment through protein fusions and interactions
C-terminus fusion N-terminus fusion SunTag GFP11

Antibody GFP1–10

Cas Cas Cas Cas

Peptide epitopes GFP11

b Effector protein recruitment through protein-RNA interactions

Stem loop modification

3′-end modification

RNA aptamer RNA interacting protein Effector protein

Examples: Examples: Examples: •Methyltransferase

•PP7 •BoxB •PCP •MCP •Activator •Histone modification protein
•Com •MS2 •Com •λ N22 peptide •Repressor •Biotinylated protein and nucleic acid
•Streptavidin •Deaminase •Florescent protein

Fig. 2 | Repurposing CRISPR as a recruiting platform. a, Effector protein recruitment through protein fusions and interactions. Effector proteins can
be fused to the C- or N-terminus of Cas. In the SunTag system, a repeat of epitopes is fused to the Cas protein, which is bound by antibodies fused to
effectors. In the self-complementing split fluorescent protein system, the 11th β-strand of GFP (GFP11) can be tandemly arrayed to recruit the rest of the
fluorescent protein (GFP1–10) fused to effectors. b, Effector protein recruitment through protein–RNA interactions. Protein-binding RNA aptamers (such
as PP7, MS2, Com and BoxB) can be incorporated into the upper stem, the first and second hairpins, and the 3′-end of the sgRNA. In the CRISPR–Sirius
system, an array of aptamers are added into the upper stem loop of the sgRNA. In the TREE system, the repeat of epitopes used in SunTag is recruited
through sgRNA.

One of the most significant advantages of the CRISPR system
is its flexibility for multiplexing, typically achieved by simultane-
ous delivery and expression of multiple gRNAs. It is possible to
deliver many gRNAs through ribonucleoproteins (RNPs) or particle
bombardment. However, since most plants rely on Agrobacterium-
mediated transfer DNA (T-DNA) transformation, the development
of efficient multiplexing expression systems is required. The most
straightforward approach is to stack multiple gRNA expression
units where each unit contains a promoter and a terminator24,115–117.
This approach can result in large constructs with many repetitive
elements, which increases cloning difficulty while decreasing trans-
formation efficiency. Therefore, multiple alternative strategies have
been developed to achieve multiplexed genome editing in plants
(Fig. 3b). One strategy is to harness the native CRISPR array expres-
sion system62,118. This is the most compact expression system and
has been successfully demonstrated in plants using Cas9 (refs. 112,119)
and Cas12a70. Notably, Cas9 CRISPR arrays may contain tandem
sgRNAs112,119 or only crRNA, with the tracrRNA expressed sepa-
rately4. Another strategy is to use the ribozyme–gRNA–ribozyme
(RGR) system in which the gRNA is flanked by a hammerhead (HH)
ribozyme and a hepatitis delta virus (HDV) ribozyme sequence108.
Tandemly arraying the RGR units allows the expression of a single
transcript followed by the precise processing of each unit, as dem-
onstrated in rice120. The third strategy is the polycistronic T-RNA–
gRNA system in which T-RNA is fused to each gRNA in a single
transcript unit, and the endogenous RNaseZ and RNaseP recognize
and cut out the T-RNAs after transcription, allowing for the forma-
tion of each mature gRNA121. This T-RNA system has been used
in many species with Cas9, including rice121, wheat (Triticum aes-
tivum)122 and maize123. The fourth strategy is the Csy4-based exci-
sion system where sgRNAs or crRNAs are flanked by Csy4 excision
sites and processed by the Csy4 endonuclease111,124. Furthermore, a
Drosha-mediated multiplexing system using sgRNA–short hairpin
RNA (shRNA) has been developed for genome editing in mamma-
lian cells125, but has not yet been demonstrated in plant cells. The
aforementioned efficient multiplexing systems can be used to target
multiple genes and gene families simultaneously for knockout, base
editing and regulation as well as large DNA fragment deletion, gene
insertion and replacement.
Precise genome editing by base editors
Base editing, using cytidine base editors (CBE) and adenine base
editors (ABE), refers to programmable and irreversible conversion
of one target nucleotide into another without DSBs or a donor tem-
plate126–128. The first generation of CBE (BE1) was engineered by fus-
ing cytidine deaminase rat apolipoprotein B mRNA editing enzyme
(rAPOBEC1)129 with dCas9 (ref. 126). Cytosine (C) deamination con-
verts C to uracil (U) which is recognized by cell replication machin-
ery as thymine (T), resulting in C-to-T transition126. However, the
conversion of C-to-U is usually reversed by base excision repair
where the U is transformed to an apurinic/apyrimidinic (AP) site
by DNA glycosylases, such as uracil DNA glycosylase (UDG)130.
To increase editing efficiency and purity, the second generation of
CBE (BE2) incorporates a uracil DNA glycosylase inhibitor (UGI)
into BE1 to block UDG126,131, causing a shift to the mismatch repair
(MMR) pathway. Editing fidelity and efficiency can be enhanced

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Review Article NATURe PlAnTS

a
CRISPR–Cas expression systems
Mixed dual promoter system
Pol II promoter Cas Terminator Pol II promoter gRNA Terminator
Dual Pol II promoter system
Pol II promoter Cas Terminator Pol II promoter gRNA Terminator
Single bidirectional promoter
Terminator Cas Bidirectional promoter gRNA Terminator

Single transcript unit system

Single promoter
Pol II promoter Cas PolyA gRNA Terminator
Single promoter with an intron
Pol II promoter Exon Intron gRNA Intron Exon Cas Terminator

b
Multiplexed gRNA expression systems
CRISPR array 6/12 nt linker 6/12 nt linker
Cas9 sgRNA
Promoter gRNA Scaffold gRNA Scaffold gRNA Scaffold Terminator
RNase III RNase III

Cas9 crRNA and tracrRNA

Promoter gRNA Scaffold cRNA Scaffold cRNA Scaffold Terminator Promoter tracrRNA
RNase III RNase III

Cas12a crRNA
Promoter Scaffold cRNA Scaffold cRNA Scaffold cRNA Scaffold Terminator

Cas12a Cas12a Cas12a

Polycistronic T-RNA–gRNA
Promoter T-RNA gRNA Scaffold T-RNA gRNA Scaffold T-RNA gRNA Scaffold Terminator
R

R
R

R
N

N
N

N
as

as

as
as

as

as
e

e
e

e
P

P
Z

HH–gRNA–HDV
Promoter HH gRNA Scaffold HDV HH gRNA Scaffold HDV HH gRNA Scaffold HDV Terminator

Cys4 system
Promoter Cys4 site gRNA Scaffold Cys4 site gRNA Scaffold Cys4 site gRNA Scaffold Cys4 site Terminator Promoter Cys4

Cys4 Cys4 Cys4

Drosha-mediated gRNA–shRNA
Promoter gRNA Scaffold shRNA gRNA Scaffold shRNA gRNA Scaffold Terminator
Drosha Drosha Drosha Drosha

Fig. 3 | Diverse CRISPR expression and multiplex systems. a, CRISPR–Cas expression systems. Cas and gRNA can be expressed under mixed dual
promoters, dual RNA Polymerase II (Pol II) promoters, a single bidirectional promoter and as a single transcript unit. b, Multiplexed gRNA expression
systems. Multiplexed gRNA can be expressed using CRISPR array, polycistronic T-RNA–gRNA, HH–gRNA–HDV, Cys4 and drosha-mediated
gRNA–shRNA systems.

with the addition of more free or fused UGIs, further demonstrating
its importance132,133. Later generations of CBE (BE3 and BE4)126,132 use
nCas9 with the D10A mutation to activate MMR and induce cells to
repair the non-edited strand using the edited strand as a template.
Further optimization of BEs includes the fusion of the Gam protein
from bacteriophage Mu, which binds to the ends of DSBs and pro-
tects them from degradation, thus reducing indel formation during
base editing132. Efficiencies of base editing can be also improved by
linker modification132, codon optimization and the incorporation of
additional nuclear localization sequences134. The most commonly
used cytidine deaminases include APOBEC1 (ref. 126), activation-
induced cytidine deaminase (AID)135, Petromyzon marinus cytosine
deaminase 1 (PmCDA1)127 and APOBEC3A136,137. Since no natu-
rally known enzymes deaminate adenine (A) in DNA, E. coli TadA

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NATURe PlAnTS
(ecTadA) evolved to catalyse adenine deamination in ssDNA128. In
addition, many variants of CBE and ABE have been developed to
overcome limitations of current base editors, thus expanding the
toolbox for base editing. Diverse base editors and their composi-
tions, editing windows, types and efficiencies have been compre-
hensively reviewed recently138,139.
Base editors have been optimized and utilized in many plant
species and, presently, the most widely used base editor is the cyti-
dine base editor BE3 (refs. 140–144). In addition, many other base
editing systems have been demonstrated in plants. A cereal plant
codon-optimized APOBEC3A is able to generate C-to-T conver-
sions efficiently in wheat, rice and potato (Solanum tuberosum)145.
SpCas9 and SaCas9 variants have also been incorporated into BEs
to expand the targeting scope in plants140. A human AID variant
lacking a nuclear export signal has been successfully used for rice
base editing, both with and without a UGI (rBE9 and rBE5, respec-
tively)146. A PmCDA1-based CBE termed Target-AID has resulted
in efficient editing in rice and tomato147. A separate study has shown
that PmCDA1 has higher base editing activity in rice than rAPO-
BEC1111. ABEs have also been demonstrated in rice140,148, Arabidopsis
and rapeseed (Brassica napus) protoplasts149.
In addition to DNA base editing, RNA base editing offers a prom-
ising tool for post-transcriptional regulation. Currently, RNA base
editors utilize the adenosine deaminase acting on RNA (ADAR) to
convert A to inosine (I)150, which are guided by antisense oligoribo-
nucleotides151 or the Cas13 system102,103. Application of RNA base
editors in plants has not yet been reported.
Optimization of base editing is still warranted to minimize or
eliminate off-target effects152, confer more flexible targeting win-
dows and increase editing activity. Engineering of novel base editors
would offer base changes in addition to C-to-T and A-to-G transi-
tions. Recently, a hypothetical model for a transversion (CG-to-GC)
base editor has been proposed by using an AID–UDG–enCas9–
Rev1* (where Rev1* indicates evolved Rev1 with PolI-like 5'-to-3'
exonuclease activity and desired processivity) fusion protein153,
which could potentially further revolutionize base editing.
Gene replacement and insertion
Although base editing allows the conversion of nucleotides within
a small window of plant genomes, larger, precise genomic changes,
such as gene replacements and targeted gene insertions, cannot be
achieved through base editing. HDR, also known as gene targeting
(GT), is the major approach to introduce foreign genes or alter gene
contents15. While GT in higher plants was extremely difficult for
decades, the utilization of SSNs has made GT much more acces-
sible25,154,155. When SSNs are transformed into plants to create DSBs,
DNA fragments harbouring desired sequences can be introduced as
the donor templates to repair the DSBs. After DSB induction, gene
replacements or insertions can be achieved through one of the fol-
lowing pathways: canonical NHEJ (cNHEJ), microhomology-medi-
ated end-joining (MMEJ) or HDR156. Cas9 and Cas12a have been
widely used to generate DSBs and co-introduced with repair tem-
plates for precise genome editing through HDR. Given HDR-based
gene replacement is essential but challenging for precise genome
editing, more efforts have focused on improving HDR over utilizing
cNHEJ and MMEJ for gene replacement and insertion.
Improving HDR through donor design. Using a donor format
can influence the rate of HDR and editing outcomes. Donor tem-
plates can be delivered as dsDNA, such as PCR products, linear-
ized or non-linearized plasmids or ssDNA. In mammalian systems,
PCR products and ssDNA outperform supercoiled plasmids in
total knock-in efficiency, while ssDNA induces less off-target and
undesired integration than using dsDNA as the donor157. Notably,
Cas9-induced DSB repair through HDR with single-stranded oligo-
deoxynucleotide (ssODN) is bias to use donor DNA complementary
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Review Article
to the non-target strand, probably due to asymmetrical Cas9 dis-
sociation158. In general, longer homology arms promote HDR effi-
ciency159. Symmetric and asymmetric donor templates may have
different effects on knock-in efficiencies157,158. Interestingly, the
gRNA-to-mutation distance may affect the efficiency and the zygos-
ity of HDR consequences157. With limited HDR research in plants, it
would be useful to compare donor types as well as lengths and posi-
tions of homology arms to improve HDR in plant cells.
Another approach to increase donor availability is to sim-
ply increase the amount of donor DNA delivered to the cell. In
plants, when CRISPR reagents are delivered into cells through
Agrobacterium-mediated transformation, donor DNA fragments
can be flanked by the target sequences so that even if the donors
are integrated into the genome, they can be liberated by the nucle-
ase160,161. This method is considered much more efficient than using
a circular plasmid162. To increase donor amount, free dsDNA or
ssDNA donors can be delivered alongside the CRISPR reagents
through particle bombardment155,163. A geminivirus-based rep-
licon system can be used to amplify DNA donor in plant cells.
Initially delivered through Agrobacterium, the geminivirus system
allows rolling circle replication of the donor to increase HDR effi-
ciency154,164–166. With the development of CRISPR delivery methods,
more approaches, such as nanoparticles, will be available to deliver
large quantities of DNA donors167,168.
Direct recruitment of donor DNA to the target site represents
another effective approach for improving HDR. One strategy is to
facilitate the transportation of the donor sequence into the nucleus.
The 5′-end of donors can be modified by adding an NLS peptide
through the interaction between an RNA adaptor and a peptide
nucleic acid, which increases donor potency and HDR efficiency169.
Additionally, biotinylated donor DNA can be recruited to tar-
get sites through streptavidin–biotin interactions, where an S1m
aptamer is added to the SpCas9 sgRNA stem loop to recruit strep-
tavidin97. Similarly, avidin can be directly fused to Cas9 to recruit
biotinylated donor DNA, thus increasing local donor availability170.
More recently, a new covalent tethering method has been developed
through a fusion between HUH endonucleases and Cas9 (ref. 171).
When the recognition sequence is added to the ssODN, it can cova-
lently bind to the HUH endonucleases and increase HDR efficiency
up to 30-fold171. While no similar studies have been reported in
plant cells, these targeted donor recruitment strategies represent
promising avenues for achieving high HDR efficiency in plants.
Interestingly, RNA molecules highly expressed from a DNA tem-
plate may also serve as donors for HDR in yeast172 and plants173,
albeit with relatively low efficiencies, encouraging further explora-
tion of using RNA donors to improve HDR in plants.
Improving HDR by modulating repair pathways. HDR enhance-
ment can be achieved by overexpression of key enzymes involved
in this pathway. For instance, RS-1 treatment and RAD51 mRNA
injection have been shown to enhance Cas9- and TALEN-mediated
knock-in efficiency in rabbit embryos174,175. In plants, it has not been
thoroughly investigated whether manipulating RAD51 expression
can increase DSB-induced HDR. With a better understanding of
plant HDR pathways, more enzymes involved in DNA repair, such
as RAD54, RAD50, CAF1 and SMC6B (also known as MIM), may
be manipulated to enhance DSB-induced HDR efficiency176,177.
HDR can also be promoted by using a nCas9 (ref. 158) since
single-stranded breaks induced by the nickase play a critical role
in promoting the HDR pathway. Moreover, nicks are not the sub-
strates of the competing NHEJ pathway, which is usually triggered
by DSBs. An in trans-paired nicking system has been developed
to introduce nicks in both targeted genomic DNA and the donor
plasmid to achieve seamless homology-directed gene inser-
tion at a higher efficiency than using Cas9 nuclease178. nCas9-
induced HDR has also been successfully demonstrated in plants25.
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Alternatively, NHEJ pathways can be inhibited genetically or
chemically. In mammalian cells when DNA LIGASE 4 (LIG4)
is inhibited by RNA interference, SCR7 or the adenovirus 4
E1B55K and E4orf6 proteins, HDR efficiency can be signifi-
cantly increased179,180. This also holds true when Ku70 and Ku80
have been knocked down179. Two DNA-dependent protein kinase
catalytic subunit inhibitors, NU7441 and KU-0060648, have been
demonstrated to reduce NHEJ-mediated repair while increasing
HDR-mediated repair181. In plants, HDR enhancement has been
observed in Arabidopsis lig4 and ku70 mutants182 as well as in rice
lig4 mutants183. While it is not desirable to use NHEJ mutants, it is
feasible to promote HDR with simultaneous knockdown of NHEJ
genes by RNA interference or CRISPR interference (CRISPRi),
which will be introduced in the next section.
Transcription regulation and epigenome editing
CRISPR–Cas can be used to alter gene expression by editing regula-
tory elements, including promoters, transcription factors, enhanc-
ers and so on. For instance, editing regulatory elements can result in
a wide range of expression levels for a gene of interest, allowing for
the discovery and selection of novel alleles, dissecting the function-
ality of gene regulatory elements and manipulation of quantitative
trait loci184,185. In addition, editing splicing sites can alter the expres-
sion of different gene isoforms, enabling the discovery of splice vari-
ants and splicing mechanisms149,186,187.
CRISPR–Cas can also be engineered for transcriptional regula-
tion. Gene knockdown was first demonstrated with CRISPR–dCas9
by inhibiting the initial binding of the transcription machinery when
targeting the promoter region or blocking RNA polymerase elonga-
tion when targeting the coding region31. Repression efficiency can
be improved by using CRISPR to recruit repressors, such as SRDX
in plant cells115,188. These techniques are collectively referred to as
CRISPRi. In contrast, when activators are recruited through CRISPR,
endogenous genes can be overexpressed in their native environment,
which is termed CRISPR activation. Activators that have been used
for this purpose include the transactivation domain of the nuclear
factor-κB p65 subunit (p65AD), the herpes simplex viral protein
16 (VP16) and its tandem versions (VP64, VP128 or VP160)189,
Epstein–Barr virus R transactivator (RTA)190, p300 (ref. 191),
activation domain from human heat-shock factor 1 (HSF1)91, tran-
scription activation domain (TAD) from Xanthamonas transcrip-
tion activator-like effectors (TALEs) as well as plant activators EDLL
and ERF2m (modified ERF2)192. Tethering multiple types of effector
proteins to the target site through prementioned protein recruiting
systems (Fig. 2) can dramatically augment transcriptional regula-
tion. For instance, successful activation has been achieved through
VP64-p65AD-Rta (VPR)190, synergistic activation mediator (SAM)91,
SunTag89,191,193 and TREE systems100. Moreover, gene regulation is
also affected by the position of the target site194. Another route to
further modulate expression is to use multiple gRNAs to target one
gene, maximizing the accessible effector proteins195,196. Interestingly,
gene repression and activation can be achieved at the same time
using dead Cas activators targeting different sites197, which allows
for the manipulation of more complex pathways. Furthermore,
since a shortened gRNA allows Cas binding without cleavage,
programming gRNA length serves as an elegant strategy to switch
between genome editing and transcriptional regulation with either
Cas9 (ref. 198) or Cas12a199.
CRISPRi and CRISPR activation technology also hold great
promise in plant transcriptional regulation66,115,188. Tethering activa-
tors through protein recruiting systems, such as MS2–MCP92 and
SunTag193, have been shown to significantly improve plant transcrip-
tional activation. A 6TAL–VP128 (TAL refers to TALE TAD motif)
activation system has been demonstrated in plants that outperforms
VP64 when delivered by Cas9 fusion or MS2–MCP recruitment,
providing a highly efficient platform for plant gene upregulation192.
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However, more research is still needed for plants regarding the dis-
covery of new regulators, optimization of targeting sites, develop-
ment of tunable and inducible regulation, and large-scale regulation.
Transcription can also be controlled by editing epigenetic marks.
Methyltransferases and acetylases can be recruited to alter the epi-
genetic modifications associated with regulation200–203. In plants,
DNA methylation193 and demethylation204 have been successfully
achieved using SunTag systems to silence and upregulate gene
expression, respectively. However, genome-wide hypermethylation
has been observed in plants193 and mammalian systems205 when
using CRISPR-directed methyltransferases, raising potential con-
cerns when using this tool for epigenome editing.
Gene expression levels can be regulated at the post-transcrip-
tional level by RNA-targeting CRISPR systems such as Cas13 and
RNA-targeting Cas9s. Moreover, SpCas9 can target RNA when
presented with a PAM sequence on a separate oligonucleotide
(PAMmer) alongside a gRNA206. In addition to SpCas9, dCas9 fused
with an RNase can target RNA for degradation207. Furthermore,
RNA sequences can be precisely changed with RNA base editing103.
Plant genome editing challenges and opportunities
The diverse CRISPR toolbox and advanced technologies provide
great platforms for plant genome engineering. However, challenges
still remain in the application of genome editing tools in plants.
With the development of new technologies and knowledge, these
challenges could be overcome.
Temperature sensitivity of Cas9 and Cas12a. Cas9 and Cas12a
require higher temperatures to achieve optimal editing efficiency
in mammalian cells208, zebrafish209 and plants210,211. In Arabidopsis,
repeated high temperature treatments result in a dramatic increase
of Cas9 efficiency212. Likewise, heat treatments result in effi-
cient Cas12a editing in Arabidopsis (29 °C) and maize (28 °C)211.
Therefore, it is important to consider temperature when applying
these CRISPR–Cas systems in plants.
Generation of transgene-free edited plants. When CRISPR is used
for crop improvement, it is desirable to obtain final products without
transgenes to minimize regulatory burden, foster public acceptance
and mitigate potential ecological consequences. CRISPR transgenes
may be eliminated through breeding and screening the segregat-
ing populations, which can be aided by coupling CRISPR-encoding
genes with fluorescent markers or herbicide susceptibility213,214.
However, this approach is not feasible for vegetatively propagated
plants or plants with long life cycles, such as trees. It could be even
more complicated for self-incompatible and polyploid plants.
Another approach to achieve transgene-free genome editing is
to generate mutated plants without transgene integration. DNA
or RNA encoding the CRISPR machinery can be introduced into
plants and transiently expressed to generate edits215,216. In addition,
CRISPR–Cas RNPs can be delivered into plant protoplasts through
polyethylene glycol (PEG)-mediated transformation, which has
been demonstrated in Arabidopsis, wild tobacco (Nicotiana attenu-
ata), lettuce (Lactuca sativa), rice74,217, apple (Malus pumila), grape
(Vitis vinifera)218, Petunia × hybrida219 and soybean74. However,
regeneration of genome-edited plants from protoplasts poses a
huge challenge for many species and could potentially introduce
unwanted somaclonal variations derived from the lengthy tissue
culture process220. Alternatively, RNPs can be delivered into plant
tissues that are routinely used for plant regeneration (such as cal-
lus) by particle bombardment221,222. RNPs have been delivered into
plant zygotes produced by in  vitro fertilization, which were later
used to regenerate mature plants without selection223. RNP delivery
eliminates the introduction of foreign DNA and shortens the expo-
sure time of genomic DNA to CRISPR reagents, hence minimizing
random DNA integration and off-targeting effects in the genome.
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NATURe PlAnTS Review Article
a b c d e f
CRISPR with BBM and WUS Asexual propogation Simultaneous gene editing and Domestication Cas or dCas–spo11 Allele stacking
(co-transformed/pre-transformed) haploid induction by CRISPR promotes meiotic
recombination
2n 2n × 2n
C D
2n × 2n B
A
Gamete Elite line Haploid inducer Elite
mother cell line with CRISPR Cas or dCas– gRNA alleles
Callus Meristem reagent Spo11 lines lines
induction MiMe by CRISPR

Chromosome shuffling
and editing
Wild ancestor

by CRISPR
2n

Domestication
n

by CRISPR
2n

Haploid seeds with edits

Gamete
Subculture 2n
BBM1 Chromosome
MTL doubling
expression
mutation D
in egg cell
C
B
A
Elite
2n 2n alleles
2n 2n

Accelerated tissue culture- Targeted chromosomal Breeding without

dependent and -independent Generation of transgene-free Domesticated breaks promote segregation of
genome editing Maternal clone generation homozygous-edited elite lines crop recombination elite alleles

Fig. 4 | Revolutionizing plant breeding by combining CRISPR with other cutting-edge technologies. a, Transformation efficiency can be elevated
by co-transforming or pre-transforming BBM and WUS2 genes. Genome-edited plants can be generated through tissue culture-dependent regeneration
or tissue culture-independent meristem induction and subculture. b, Asexual propagation of genome-edited plants can be achieved by creating the
genotype MiMe followed by haploid introduction through ectopic expression of BBM1 in the egg cell or knocking out MTL. c, Simultaneous gene editing and
haploid induction can be achieved by crossing elite lines with haploid inducer lines carrying genes encoding the CRISPR system. Edited haploids
without CRISPR genes can be selected and used to make transgene-free double haploid lines. d, De novo domestication of crops can be achieved using
CRISPR in wild species, resulting in improved crops with desirable traits. e, Cas or dCas–spo11 can be used to promote meiotic recombination to break
tight linkage and facilitate breeding. f, Elite alleles located at different chromosomes can be stacked at one locus by CRISPR to avoid segregation
during breeding.

Recently, a transgene killer CRISPR system has been developed to
link suicide transgenes with CRISPR constructs and eliminate all
transgenes in the genome of edited T1 rice plants224.
Genome editing of polyploid plants. Polyploid plant species make
up a significant portion of major crops, including staple food, fruit
and industrial crops. Commonly planted and consumed polyploid
crops include triploids (citrus, banana (Musa acuminata, M. balbisi-
ana and Musa × paradisiaca), seedless watermelon (Citrullus lana-
tus) and some varieties of apples); tetraploids (pasta wheat (Triticum
durum), potato, cotton, canola (B. napus), rapeseed, peanut (Arachis
hypogaea), tobacco, switchgrass (Panicum virgatum) and some vari-
eties of apple); hexaploids (Camelina sativa, bread wheat and oats
(Avena sativa)); and octoploids (sugar cane (Saccharum officinarum)
and strawberry (Fragaria × ananassa)). Gene knockout efficiency is
usually lower in polyploid plant species than diploids, as multiple
alleles must be edited simultaneously. Key factors for successful
polyploid plant genome editing include an efficient expression sys-
tem and a highly active Cas nuclease. To date, multiallelic genome
editing has been achieved in several polyploid plant species, includ-
ing both model systems225,226 and crops221,227–236. Successful gene edit-
ing has been used to introduce valuable agronomic traits such as
improved oil quality230 and disease resistance233,237. Many attempts
have even been made to produce transgene-free edited polyploid
plants through RNP delivery221 and selection-free methods234.
Although both approaches are labour- and time-consuming, they
offer the potential to accelerate applied research in crops. However,
in addition to gene knockout, precise genome editing through HDR
in stable transgenic lines of polyploid plants remains challenging.
Besides crop improvement, genome editing in polyploid plants
also offers a platform for gene dosage studies in which edited lines
with varying copy numbers of functional genes can be obtained
in one round of transformation and plant regeneration225,232. This
allows for quantitative studies of the relationship between genotype
and phenotype.
Germline editing by floral dip transformation. Generation of
germline-edited plants is important for downstream genetic and
trait analysis. Ironically, this remains a challenge for plants where
the convenient Agrobacterium-mediated floral dip method is used
to deliver CRISPR transgenes, such as Arabidopsis, not only due to
low editing efficiency in germlines24,238,239, but also the dissociation
of editing results in somatic and germinal cells240. Agrobacterium
is thought to deliver CRISPR-carrying T-DNA into the egg cells.
Germline edits can be obtained only if CRISPR makes edits after
Agrobacterium infection but before the first embryogenic cell divi-
sion. Edits occurring afterwards will most likely result in chimeric
plants. To overcome this challenge, tissue-specific promoters have
been used for Cas9 expression to limit or boost genome editing in
germinal cells, including egg cell-specific promoter EC1.2 (ref. 26),
sporogenesis expression promoter SPOROCYTELESS241 and meio-
sis I-specific promoter242, as well as CDC45, DMC1, SOP11, YAO
and RPS5A promoters, which are preferentially expressed in actively
dividing tissues243–246. HDR was successfully achieved in Arabidopsis
when a sequential transformation method was used, which takes
advantage of preselected transgenic lines with high germline expres-
sion of Cas9 (ref. 247). While the emerging oil crop Camelina faces
similar problems to Arabidopsis, it remains to be seen whether any
of the aforementioned strategies could improve genome editing in
this species230.
Off-target effects in plants. The off-target effect of Cas proteins
is usually a major concern of CRISPR technology in applied sci-
ences, especially in gene therapies. Cas9-induced DSBs could result
in large deletions and genome rearrangements248. In plants, inten-
sive investigations have been carried out to detect off-target effects
by whole genome sequencing in Arabidopsis, rice, cotton and so
on220,249,250. Studies have revealed a high specificity of genome edit-
ing in plants using wild-type Cas9 and Cas12a, showing that most
mutations found in edited plants are due to somaclonal variations.
However, whole genome sequencing has shown that CBEs, not

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Review Article NATURe PlAnTS

a b

Gene family- or Single gene Whole genome library

Single gene
Whole genome library pathway-specific regulatory element
library CRISPR–Cas
library library Protoplast
transformation CRISPRi
CRISPRa

Plant transformation with CRISPR–Cas

Reporter
cell line

Selection Cell sorting

Expression level
6
4
2
0
W

Li

Li

Li
ne

ne

ne
T

Identify essential Study biological Study gene function Study regulatory Identify genes of interest by sequencing
genes problems and evolve genes elements

Fig. 5 | Genetic screens with CRISPR libraries in whole plants and plant cells. a, Genetic screens with CRISPR libraries in whole plants. gRNA libraries
targeting a whole genome, a gene family or pathway, a single gene coding sequence or regulatory elements can be transformed into plants along with
a chosen Cas nuclease or base editor. Collections of mutants can be generated to identify essential genes or study functionality of genes, regulatory
elements or in planta gene evolution. b, Genetic screens with CRISPR libraries in plant cells. gRNA libraries targeting the whole genome can be
transformed into plant cells such as protoplasts. If traits of interest can be linked to drug-resistant selective markers, protoplasts can be used for plant
regeneration on selective media. If traits of interest can be linked to fluorescent protein expression, cells can be sorted by fluorescence-activated cell
sorting. Selected or sorted cells can be further sequenced to identify genes or regulatory elements contributing to traits of interest.

ABEs, induce substantial genome-wide off-target effects in rice152;
therefore, their use may necessitate additional screening and purify-
ing selection. Moreover, recent studies have shown that both CBEs
and ABEs induce transcriptome-wide RNA off-target modifications
in human cells251,252. However, base editors can be engineered to sig-
nificantly reduce their RNA editing activity251,252.
To minimize potential off-target mutations, paired nCas9s can
be used30. Protein engineering has been used to reduce Cas bind-
ing affinity (thus increasing editing specificity), resulting in high
fidelity versions of SpCas9 such as SpCas9-HF1-4 (ref. 253), SpCas9
(K855A) and eSpCas9 (1.1)254, HypaCas9 (ref. 255), xCas9 (ref. 34),
evoCas9 (ref. 256), sniper-Cas9 (ref. 257) and HiFi Cas9 (R691A) in
the context of RNP delivery258 (Table 1). In rice, eSpCas9 (1.0 and
1.1) and SpCas9–HF1 have been demonstrated to maintain their
on-target editing activity with enhanced specificity when using the
T-RNA–sgRNA processing system259. Genome editing with two
Cas9 variants, eHF1-Cas9 and eHypa-Cas9, have also been dem-
onstrated in rice260. Recently, xCas9 was shown to have a higher
targeting specificity than wild-type Cas9 in rice40. However, owing
to intrinsically lower nuclease activities for many high-fidelity
SpCas9s in plants259,261, they may not be widely adopted for plant
genome editing before further improvement. Other approaches to
minimize off-target effects include designing gRNAs with fewer
potential mismatch targets in a given genome. Additionally, limit-
ing the exposure of the genome to CRISPR reagents, for example,
through transient expression and RNP transformation, can reduce
the potential for off-target activity.
Although it is hard to eliminate the possibility of CRISPR-
induced large chromosomal deletions, insertions, inversions
and rearrangement in plant genomes, which are often difficult to
detect with commonly used sequencing technologies, the off-target
effects should not be an obstacle in plant genome editing based on
the knowledge generated so far. One can not only avoid off-target
mutations during the process of generating genome-edited plants,
but also detect and screen out products with off-target mutations
through breeding and selection, if necessary.
Revolutionizing breeding by combining CRISPR with other
cutting-edge technologies. Generating uniform plants harbour-
ing CRISPR-induced edits relies heavily on tissue culture-based
embryogenesis or organogenesis, especially for many major crops.
This limits the application of CRISPR in recalcitrant or marginally
transformable plant species and varieties. One approach to overcome
this difficulty is to express plant growth-stimulating genes to boost
plant transformation and regeneration. With the overexpression of
transcription factors Baby boom (BBM) and Wuschel2 (WUS2), pro-
found improvement of transformation efficiency was achieved in
recalcitrant maize lines, sorghum (Sorghum bicolor), sugarcane and
indica rice262,263. Furthermore, BBM, WUS or similar factors may
even allow tissue culture-free genome editing (if they are induced
or applied ectopically) alongside genome editing reagent delivery
to stimulate meristem tissue formation from somatic tissues, repre-
senting a novel and effective approach to achieve genome editing in
many difficult plant species (Fig. 4a).

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NATURe PlAnTS
In plant breeding, it is preferable for desirable traits to be sta-
bly inheritable through generations. This is a big challenge for
hybrid crops benefiting from heterosis as well as self-incompatible
crops, since seed production will inevitably cause segregation. With
CRISPR, asexual propagation of seeds has been achieved by creating
the genotype MiMe (mitosis instead of meiosis) followed by hap-
loid introduction264,265. By knocking out three genes in Arabidopsis
(atosd1 atrec8 atspo11-1 triple mutants)266 and rice (ospair1-4
osrec8-3 ososd1-1 triple mutants)267, meiosis can be substituted by
mitosis to produce diploid gametes that are genetically identical to
the parent. Haploid seeds can then be induced through multiple
approaches to produce clonal progeny, including using haploid
inducer lines for crossing, ectopic expression of BBM1 (a BBM-like
gene expressed in sperm cells) in the egg cell264 as well as knock-
ing out the MATRILINEAL (MTL) gene265. CRISPR has facilitated
the development of this new technology and allows for the stable
inheritance of elite lines without the risk of segregation (Fig. 4b).
To obtain transgene-free homozygous genome-edited germ-
lines without tissue culture, two similar systems have been devel-
oped using haploid induction-edit268 and haploid-inducer mediated
genome editing (IMGE)269 (Fig. 4c). Haploid inducer lines are used
to cross with the elite lines. The genome that will not pass to the hap-
loid progeny carries the CRISPR–Cas transgene, which allows for
the elimination of the transgene in the edited plants. Edited haploids
can be selected and subsequently used to make double haploid lines.
Without any doubt, plant breeding will further benefit from
CRISPR technology. Recently, several studies have demonstrated
the power of CRISPR in de novo plant domestication270–272 (Fig. 4d).
In the future, CRISPR will speed up the plant breeding process when
coupled with new ideas. For instance, Spo11, involved in generating
DSBs for meiotic recombination, can be recruited through dead Cas
to break tight linkage among genes, which can help overcome link-
age drag in breeding273 (Fig. 4e). Moreover, CRISPR can be used to
achieve chromosome shuffling to stack multiple elite alleles into one
tightly linked locus, thus preventing the segregation of elite alleles
during breeding (Fig. 4f). With CRISPR genome editing tools, plant
breeding will be elevated to a whole new era in a more precise and
customized manner, which cannot be achieved through conven-
tional breeding and traditional genetic engineering.
Prospects
The rapid development of CRISPR technology will facilitate numer-
ous applications in plant biology. For instance, the use of gRNA librar-
ies will enable large-scale screening to discover the functions of genes
and regulatory elements (Fig. 5a). With Agrobacterium-based T-DNA
delivery, pooled CRISPR libraries were used to generate a collection
of mutants for gene families in tomato274 and nearly all coding genes
in rice275,276, providing valuable genetic resources for basic research
and breeding. The scale can be shrunk to target a single gene, tilling
the coding sequencing for in planta gene evolution277 or targeting reg-
ulatory elements to generate quantitative trait variation184. However,
it is time- and labour-consuming to deliver CRISPR libraries into
plants for downstream applications. In the future, we envision the
possibility to deliver gRNA libraries into plant cells (such as proto-
plasts) to empower cell-based screens. By linking traits of interest to
selectable markers, such as drug resistance and fluorescent protein
expression, individual plant cells can be selected or sorted for analysis
and regeneration (Fig. 5b). This will present an exciting opportunity
to link genotype to phenotype in a high-throughput manner.
Plants also store genetic information in two organelles: mito-
chondria and plastids. Organelle genome editing could be used to
manipulate metabolic pathways to gain desired phenotypes. In gen-
eral, mitochondria can repair DSBs through both HDR and NHEJ
pathways278. However, in mammalian cells, mitochondria cannot
efficiently repair DSBs, leading to the degradation of mitochondrial
DNA (mtDNA) and a shift of the heteroplasmic ratio279,280. Genome
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Review Article
editing in mammalian mitochondria has been achieved using
TALENs281 and ZFNs282. In order to implement CRISPR in mito-
chondria, many attempts have been made to import RNA into mito-
chondria with little success283. One study showed mtDNA editing
using mitochondria localized Cas9 and normal gRNAs, suggesting
that Cas9/gRNA RNPs might be preassembled before transpor-
tation into mitochondria284. In plastids such as chloroplasts, the
NHEJ repair pathway is thought to be absent278. While chloroplast
genomes can be edited through HDR without the use of any SSNs285,
it is likely that HDR in plastids can be improved by introducing tar-
geted DSBs by SSNs, such as CRISPR. Currently, genome editing in
either plant mitochondria or plastids has not been achieved using
SSNs, including CRISPR. However, a recent report on efficient chlo-
roplast transformation in CRISPR-generated Arabidopsis mutants
(more sensitive to the selection agent) has provided more opportu-
nities in this exciting research area286.
Although components of genome editing reagents can be well
designed, the editing outcomes, which highly rely on DNA repair
choices, cannot be strictly controlled thus far. Edits generated by
cNHEJ and MMEJ pathways are strongly correlated with the target
sequence context287,288 and the broken ends generated by SSNs289,290.
Through editing data profiling, machine learning and modeling,
deletion and insertion patterns as well as probabilities can be pre-
dicted287,291,292. With a thorough understanding of editing results,
gRNA can be better designed to achieve diverse goals. With the
differences in DNA repair pathways and preference, the prediction
models and algorithms developed in certain types of human cells
may not be readily applicable to a specific plant species. This calls
for future studies to understand DNA repair pathways in plants to
better predict and manipulate editing outcomes.
CRISPR is not only a platform for genome editing and genetic
manipulation, but also serves as a new avenue to localize defined
effector molecules (for example, protein, RNA or DNA) to specific
genomic and transcriptomic loci, allowing CRISPR to be repur-
posed for many diverse applications (Fig. 2), such as visualizing
specific regions of DNA and RNA by recruiting fluorescent pro-
teins293–295, and purifying and isolating DNA- or RNA-associated
proteins and nucleic acids296. In the future, CRISPR technology will
accelerate plant biology research, ultimately furthering the develop-
ment of new germplasm and even new crops. It will play a critical
role in addressing agricultural, environmental and ecological issues
while promoting food security in the face of climate change and
population growth.
Received: 6 April 2019; Accepted: 24 May 2019;
Published online: 15 July 2019
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Acknowledgements
Due to limited space, we could not cite all the related literature. We apologize to the
authors whose work was not cited in this Review. Our plant genome engineering research
is supported by the National Science Foundation Plant Genome Research Program (grant
no. IOS-1758745), Biotechnology Risk Assessment Grant Program (grant no. 2018-
33522-28789) from the US Department of Agriculture, the Foundation for Food and
Agriculture Research (grant no. 593603) and Syngenta Biotechnology.
Author contributions
Y.Z., A.A.M., S.S. and Y.Q. wrote the manuscript. Y.Z. prepared the figures. All authors
read and approved the final manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence should be addressed to Y.Q.
Peer review information: Nature Plants thanks S. Toki, L. Xia and the other, anonymous,
reviewer(s) for their contribution to the peer review of this work.
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