Plant Science 333 (2023) 111746

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Review article

Development and application of CRISPR/Cas9 to improve anthocyanin

pigmentation in plants: Opportunities and perspectives
Enerand Mackon a, Guibeline Charlie Jeazet Dongho Epse Mackon b, Yongqiang Guo b,
Yafei Ma b, Yuhang Yao b, Piqing Liu b, *
a
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, PR China
b
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, Nanning 530005, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Since its discovery in 2012, the novel technology of clustered regularly interspaced short palindromic repeats
Plant secondary metabolites (CRISPR) and CRISPR-associated protein 9 (Cas9) has greatly contributed to revolutionizing molecular biology. It
Anthocyanin has been demonstrated to be an effective approach for identifying gene function and improving some important
Natural pigments
traits. Anthocyanins are secondary metabolites responsible for a wide spectrum of aesthetic coloration in various
CRISPR/Cas9
Gene editing, anthocyanin regulation
plant organs and are beneficial for health. As such, increasing anthocyanin content in plants, especially the edible
tissue and organs, is always a main goal for plant breeding. Recently, CRISPR/Cas9 technology has been highly
desired to enhance the amount of anthocyanin in vegetables, fruits, cereals, and other attractive plants with more
precision. Here we reviewed the recent knowledge concerning CRISPR/Cas9-mediated anthocyanin enhance­
ment in plants. In addition, we addressed the future avenues of promising potential target genes that could be
helpful for achieving the same goal using CRISPR/Cas9 in several plants. Thus, molecular biologists, genetic
engineers, agricultural scientists, plant geneticists, and physiologists may benefit from CRISPR technology to
boost the biosynthesis and accumulation of anthocyanins in fresh fruits, vegetables, grains, roots, and ornamental
plants.

1. Introduction fungal disease resistance (Gadikota et al. 2001; Kangatharalingam et al.

Plant coloration is historically very important in molecular biology
since it was the first trait used for revealing basic genetic rules by
Mendel in the 19th century. Besides chlorophyll, three types of plant
pigments determine the coloration of plants, including anthocyanins,
betalains, and carotenoids (Xia et al. 2021). Anthocyanins are secondary
metabolites that belong to the flavonoid family, a sub-group of phenolic
compounds, responsible for a wide spectrum of aesthetic coloration
(vivid red, blue, and purple) in various plant organs, including flowers,
leaves, stems, shoots, fruits, and grains (Mackon et al. 2021b). Epide­
miological and preclinical trials revealed that anthocyanin is a
health-promoting compound (Kozłowska and Dzierżanowski 2021;
Mattioli et al. 2020; Salehi et al. 2020). Increased consumption of
anthocyanin-rich fruits and vegetables may aid in the prevention of
several diseases in our bodies, including cancer, cardiovascular disease,
and obesity (Cappellini et al. 2021; Khoo et al. 2017; Shang et al. 2019).
High concentrations of the pigment are associated with bacterial and
2002), heavy metals (Shoeva and Khlestkina, 2018), heat stress (Zhang
et al. 2019), and other stresses (Landi et al. 2015). Anthocyanin has also
served to trace the evolutionary history of rice (Xia et al. 2021).
Recently, anthocyanin has been revealed as a potential visible marker
employed in visualizing transgenic events in plants (He et al. 2020),
providing a useful system to study the regulation of genes in higher
plants. Because the identification of anthocyanins’ pigmentation does
not require any other supplemental treatment, they are the most easily
identified traits. As a result, the anthocyanin trait is favorable not only
for its visibility but mostly for its applicability in crop science.
Understanding the molecular basis of anthocyanin pigmentation has
been set as an important research goal during the last decade. The visual
signs of pigment deposition in tissues likely guided scientists in inves­
tigating the molecular basis of anthocyanin synthesis in model plants
like petunia and maize (Dooner et al. 1991). To date, the anthocyanin
biosynthesis mechanism has been widely studied and is relatively clear
in many species, and repressors and activators have been identified

* Corresponding author.
E-mail address: liupq@gxu.edu.cn (P. Liu).

https://doi.org/10.1016/j.plantsci.2023.111746
Received 22 February 2023; Received in revised form 22 April 2023; Accepted 21 May 2023
Available online 23 May 2023
0168-9452/© 2023 Published by Elsevier B.V.
E. Mackon et al. Plant Science 333 (2023) 111746

(Cappellini et al. 2021; Khusnutdinov et al. 2021; Xia et al. 2021). One of
the most important things to figure out right now is how to make
anthocyanin in plants more valuable from a business point of view. The
efforts have been focused on the activation of the endogenous antho­
cyanin biosynthesis genes by metabolomic engineering. For e.g Zhu
et al. engineered a novel biofortified purple endosperm rice, called
"Zijingmi," using a high-efficiency vector system incorporating two
regulatory genes and six structural anthocyanin-related genes controlled
by endosperm-specific promoters. The 13 natural anthocyanin biosyn­
thesis genes that are repressed or expressed at low levels in wild-type
rice endosperm were activated (or enhanced) by the expression of the
transgenes, which results in the formation of anthocyanin (Zhu et al.
2017). Likewise the management of environmental conditions such as
light (Huang et al. 2019a), nutrient supply (Sinilal et al. 2011;
Yamuangmorn et al. 2018), conventional breeding, and the introgres­
sion of dominant mutations through interspecific crosses with wild
species (Jones et al. 2003; Mes et al. 2008), etc were reported. But these
methods have some limitations, like taking a long time, being expensive,
not working well, being limited by reproductive isolation and the effect
of genes that aren’t linked well, and so on.(Scheben et al. 2017).
With the rapid evolution of molecular technology, the novel tech­
nique of clustered regularly interspaced short palindromic repeats
(CRISPR) is highly desirable for achieving the same goal with more
precision. Since it was discovered in bacteria and archaea at the end of
the XX century, the CRISPR system has revolutionized site-directed
mutagenesis, so-called genome editing technology (GET), during the
last decade and been recognized with a Nobel Prize recently in 2020
(Khusnutdinov et al. 2021). One of the biggest advantages of the
CRISPR/Cas9 system over other genome-editing tools like zinc finger
nucleases (ZFNs) and transcription activator-like effector nucleases
(TALENs) is that it can introduce multiple sgRNAs into cells at the same
time to create multiplex targeting mutations.
CRISPR/Cas9 genome editing technology has been widely used to
generate targeted genes modifications in more than 50 plant species
(Wang et al. 2022b), including Arabidopsis (Feng et al. 2013; Gao et al.
2016b; Gao et al. 2015; Mao et al. 2013), Tobacco (Li et al. 2013;
Nekrasov et al. 2013), rice (Miao et al. 2013; Xie and Yang, 2013; Xie
et al. 2017), Wheat (Shan et al. 2013), Soya beans (Cai et al. 2018);
grape (Wang et al. 2018), etc. Modifying a trait using CRISPR-Cas9
mutagenesis has been proven to provide advantages in identifying
gene function and crop improvement. CRISPR/Cas9 played more
obvious role in the identification of genes involved in anthocyanin
biosynthesis in plants and several studies has been reported (Table 1). In
addition, previous reviews highlighted the benefits of applying CRISPR
technology in agricultural breeding and domestication of wild species
(Zhu et al. 2020), improving grain quality (Fiaz et al. 2021), and
enhancing plant biotic and abiotic stresses (Wang et al. 2022b). In the
present review, we summarized the recent advances in
CRISPR/Cas9-mediated gene editing for improving anthocyanin in
plants, which included inducing the anthocyanin or increasing the
amount (Fig. 1). In addition, we discussed the future directions and
limitations of CRISPR/Cas9 anthocyanin enhancement in plants,
providing novel ideas to create super plants with high anthocyanin
content.
2. The basic principle of CRISPR/Cas9 in plant genome science
The CRISPR system is mainly composed of two elements, including
the CRISPR locus and the CRISPR-associated protein (Cas). Several Cas
nucleases have been reported: Cas12a (Cpf1), Cas12b (C2c1), Cas12e
(Cms1), and Cas9. According to the phylogeny and the difference in Cas
protein composition and amino acid sequence, the CRISPR-Cas system
was divided into three types, of which types I and III are the most
complex and type II the easiest and most commonly used (Makarova
et al. 2011). Cas9 is a DNA endonuclease that originated in bacteria such
as Brevibacillus laterosporus (Karvelis et al. 2015), Staphylococcus aureus
(Ran et al. 2015), Streptococcus thermophilus (Steinert et al. 2015), and
Streptococcus Pyogenus (Geng et al. 2016). The most frequently used for
Cas9 isolation is the latter one. Because the type II CRISPR/Cas system
contains a special Cas9 protein, it is then called the CRISPR/Cas9 sys­
tem. Currently, CRISPR/Cas9 is a widely adopted genome editing
technology (GET) because of its simplicity, efficiency, and versatility
(Fiaz et al. 2019). The main advantage of CRISPR/Cas9 is that the
enzyme cleaves DNA at a specific target site, which is most reliable, and
off-target sites are identified through sequence analysis (Zischewski
et al. 2017). The system is mainly composed of Cas proteins, which
contain two domains (HNH and RucV-like domains), and sgRNA, a
synthetic RNA with about 100 nucleotides guiding the Cas9 protein to a
specific target site. The sgRNA is composed of a programmable
CRISPR-RNA (crRNA), which is a 20-nt sequence located at its 5’ end
and contains a protospacer adjacent motif (PAM) recognition and
binding site of Cas9; and a fixed trans-activated crRNA (tracrRNA),
which is a 3’-end sgRNA loop structure that can anchor the target
sequence and form a complex with Cas9. A CRISPR-RNA transcript

Table 1
Previous studies on functional characterization of anthocyanin genes through CRISPR/Cas9.
Plants Gene Effect Reference

Torenia fournieri F3H Flower depigmentation (Nishihara et al. 2018)

Daucus carota F3H Change in color resulting in white callus cells (Klimek-Chodacka et al.
2019)
Solanum lycopersicum F3H depigmentation of the hypocotyl (Liu et al. 2021a)
Oryza sativa F3’H Changes of seed color and decreasing in anthocyanin content from 41.9 to 4 (Jung et al. 2019)
mg/g
Euphorbia pulcherrima (Poinsettia) F3’H the change of bract color from red to yellow (Klimek-Chodacka et al.
2019)
Ipomoea nil (Japanese morning DFR Anthocyaninless white flowers (Watanabe et al. 2017)
glory)
Oryza sativa DFR changes of seed color and decreasing in anthocyanin content from 41.9 to 4 (Jung et al. 2019)
mg/g
Solanum lycopersicum DFR the reduction of anthocyanin pigmentation in regenerated (Danilo et al. 2018)
plantlets
Gentiana scabra (Japanese gentian) Gt5GT, Gt30 GT, and Gt5/30 Anthocyanin pigmentation resulted in a specific flower color shade (Tasaki et al. 2019)
AT
Oryza sativa OsTTG1 decreased color in different organs (Yang et al. 2021)
Solanum lycopersicum SlMYBAN2 very less anthocyanin compared to wild-types (Yan et al. 2020)
Nicotiana tabacum NtAN1a and NtAN1 depigmentation of seed and flower (Tian et al. 2021)
Brassica napus BnTT8 depigmentation of seed (Zhai et al. 2020)
Oryza sativa MRP15 Leaves loss coloration from purple to green (Ma et al. 2015)
Oryza sativa OsGSTU Leaves loss coloration from purple to green (Ma et al. 2015)

2
E. Mackon et al. Plant Science 333 (2023) 111746

Fig. 1. Graphical abstract, Anthocyanin improvement in several plants through CRISPR/Cas9 GET.

Fig. 2. Scheme of anthocyanin biosynthesis pathway, storage, and its regulatory system.

3
E. Mackon et al. Plant Science 333 (2023) 111746

produces one or more short guide RNAs to direct Cas9 to the target DNA
sequence. The sgRNA/Cas9 complex is formed through Watson-Crick
base pairing after cas9 recognizes the target site. A sgRNA/Cas9 com­
plex cleaves the target DNA strand complementary to the guide RNA
sequence and the non-target strand, respectively, resulting in a double
strand break (DSB). DSBs induce DNA repair processes through either
nonhomologous end-joining (NHEJ) or homologous-directed repair
(HDR). NHEJ repairs most DSBs, creating mismatches and gene in­
sertions and deletions (indels) that cause gene knockout. HDR causes
gene substitutions or foreign DNA knock-ins using oligo-templates
(Chang et al. 2015; Jacobs et al. 2015). CRISPR/Cas9 can modify the
genomes of people, animals, and plants using DNA repair processes.
Researchers have improved the efficiency of gene knock-out through
multiple gRNAs with identical promoters (Ma et al. 2015). This process
is also employed to knock out multiple genes in the diploid and poly­
ploid genomes as well. It is now possible to construct large cassettes of
up to 24 gRNAs in plants using an advanced polycistronic gRNA
expression approach, resulting in multiplex gene editing (Hahn and
Nekrasov, 2019).
3. Genetic basis of Anthocyanin accumulation in plants
Prior to the gene editing process, the understanding of how genes
function within the pathway and the identification of possible copies are
essential. The attractive colors revealed by the anthocyanins in plants
are the consequences of complex mechanisms in which large numbers of
genes are involved. The pathway of anthocyanin biosynthesis is highly
conserved. Two main processes build the scheme of these mechanisms,
which are the synthesis and storage processes. The main step is char­
acterized by biosynthesis, which involves structural and regulatory
genes. The other step is the storage mechanism, which mainly involves
transporters.
3.1. Structural genes
The anthocyanin biosynthesis mechanism belongs to the flavonoid
branch of the general phenylpropanoid pathway, a well-studied and
characterized process in plants (Fig. 2). The process starts with malonyl-
CoA and p-coumaroyl-CoA, which are the first committed steps of the
flavonoid pathway. Under the successive catalytic activities of the early
biosynthesis enzymes (EBG), dihydroflavonols are produced. The EBGs
encode enzymes that catalyze the conversion of the precursor com­
pounds into anthocyanidin compounds. They include chalcone synthase
(CHS), chalcone isomerase (CHI), flavonoid 3’ hydroxylase and
3’5’hydroxylase (F3’ H, F3’5’H), flavone synthase (FLS), and flavone 3-
hydroxylase (F3H), known as very important genes in flavonoid
biosynthesis.
Dihydroflavonol is a colorless pigment and comprises dihy­
drokaempherol, dihydroquercetin, and dihydromyricetin, known as the
immediate precursors of anthocyanin. The downstream steps that lead
to anthocyanin are the most essential for anthocyanin biosynthesis and
involve late biosynthesis genes (LBGs). LBGs include dihydroflavonol
reductase (DFR), which catalyzes the reduction of dihydroflavonol to
leucoanthocyanin; anthocyanin synthase (ANS), and leucoanthocyani­
din oxidase (LDOX), involved in the oxidation of leucoanthocyanidin to
form anthocyanidin; then anthocyanin glycosyl-transferase (GT, UFGT)
is involved in the glycosylation of anthocyanidin to form anthocyanin
(Mackon et al. 2021a; Xia et al. 2021). The importance of these genes has
been demonstrated and is now revealed as the most popular target.
In this pathway, CHS, FLS, and DFR are special because CHS en­
hances the accumulation of flavonoids, while FLS and DFR may compete
for the same product (dihydroflavonol) to form flavonol or anthocya­
nidin. The LBGs are the most important since they are directly involved
in the formation of the anthocyanins. Basically, in different plants,
distinctive colors can be observed due to the presence of different types
of anthocyanin. Previous research revealed that over 500 anthocyanin
types may exist in nature (Avula et al. 2023; Hughes et al. 2021). These
anthocyanins are formed from 23 anthocyanidins found in nature, with
six (90% of the total) being most represented, including cyanidin (Cy)
(50%), delphinidin (Dp) (12%), peonidin (Pn) (12%), pelargonidin (Pg)
(12%), petunidin (Pt) (7%), and malvidin (Mv) (7%). These most com­
mon anthocyanidins occur as glycosides with different substitutions on
their B-ring aglycones (Avula et al. 2023). They form anthocyanin after
diverse modification processes (Mackon et al. 2021a), such as acylation,
glycosylation, and methylation, involving several enzymes such as
glucosyl-transferase (GT), rhamnosyl-transferase (RT),
methyl-transferase (MT), and acyl-transferase (AT). In a recent study,
this was partially demonstrated using CRISPR/Cas9, in which the knock
out of the anthocyanin glycosyl-transferase genes Gt5GT and Gt3’GT, as
well as the acyl-transferase gene Gt5/30AT, resulted in different colors
with unique flower shades due to the prevalence of various delphinidin
derivatives (Tasaki et al. 2019). This is important for ornamental plants
whose different colors could be valuable in the market.
3.2. Regulatory genes
From Mendel to Mcclintock and up to date, studies based on flavo­
noid pigments have resulted in some of the most significant scientific
discoveries of the past 150 years, including the earliest evidence of
transcriptional control in plants. The expression of the structural genes is

Table 2
Previous studies on identification of MBW complex in some plants.
Species MYB/bHLH/WDR Function events in plant Authors

Zea mays ZmC1/ZmR1, ZmB1/PAC1 Anthocyanins in kernels. (Cappellini et al. 2021)

Antirrhinum majus Ros1/Ros2/Ve–Del–W Anthocyanin synthesis and patterning in flowers and the plant (Lloyd et al. 2017)
body.
Petunia hybrida AN2/AN1/AN11 Anthocyanins in flowers and the plant body. (Hichri et al. 2011)
PH4/AN1/AN11 Flower color modification.
Arabidopsis PAPs–EGL3/GL3/TT8–TTG1 Anthocyanins in the plant body. (Gonzalez, 2009; Matsui et al. 2008)
thaliana
Arabidopsis TT2/TT8/TTG1 PAs in seed coats. (Baudry et al, 2004; Xu et al. 2015)
thaliana
Oryza sativa OsC1/Kala4/OsTTG1 Anthocyanin in rice caryopsis (Mackon et al. 2021a; Xia et al.
2021)
Populus nigra PtrMYB57/bHLH131/PtrTTG1 anthocyanin in leaves and stem (Wan et al. 2017)
Brassica napus BnTT18/BnTT8/BnTTG1 Anthocyanin and proanthocyanin in the seed coat (Ran, 2022)
Punica granatum PgAN1/PgAN2/PgWD40 Anthocyanin in fruit (Rouholamin et al. 2015)
Prunus persica PpMYB10-PpbHLH-PpWD40 (Cappellini et al. 2021)
Vitis vinifera vvMYBA1, VvMYBA2/VvMYC1/VvWDR Anthocyanin in fruit skin (Cappellini et al. 2021)
Malus domestica MdMYB1, MdMYBA/MdbHLH3, MdbHLH33/ Anthocyanin in fruit skin (Chaves-Silva et al. 2018)
MdTTG1
Actinidia chinensis AcMYBc123/AcbHLH42/AcWD40 Anthocyanin in the inner pericarp of red-centered kiwifruit (Wang et al. 2018)

4
E. Mackon et al.
regulated by the MBW complex, which is more important for regulating
late flavonoid biosynthesis genes than early genes (Lloyd et al. 2017; Xu
et al. 2015). Previous studies reported that structural genes are regu­
lated differently from monocot and dicot (Petroni and Tonelli, 2011;
Mackon et al. 2021a). The MBW control the LBGs and EBGs separately in
dicot plants like Arabidopsis (Gonzalez et al. 2008), whilst in monocot
like rice they function as a simple unit activated by the complex (Zheng
et al. 2019a; Sakulsingharoj et al. 2014). In the MBW complex, three
transcription factor (TF) families are found, namely MYB TFs,
basic-helix-loop basic (bHLH) TFs, and WD repeat TFs (Table 2). In black
rice (Oryza sativa), the MBW complex consists of OsC1-Kala4-OsTTG1,
which activates DFR and ANS to produce anthocyanin (Mackon et al.
2021a). In arabidopsis (Arabidopsis thaliana), the anthocyanin and
proanthocyanidin genes are activated via the combination of TT2, TT8,
and TTG1 (Baudry et al, 2004; Xu et al. 2015). The transcription factors
AN2, AN1, and AN11 constitute the MBW complex in petunias (Petunia
nyctaginiflora) (Hichri et al. 2011). Several members of the MBW com­
plex have been revealed through CRISPR-Cas9 (Table 1). A recent study
showed that the rice OsTTG1 CRISPR/Cas9 knockout mutant showed
drastically reduced anthocyanin accumulation in multiple organs and
trichome reduction in grains. OsTTG1 protein was able to physically
interact with Kala4, OsC1, OsDFR, and Rc (Yang et al. 2021). Trans­
parent testa8 (TT8) is one of the key members of the MBW complex. It
has been reported as a unique bHLH TF involved in anthocyanin
revealed through CRISPR-Cas9 editing, and most plant species have one
copy (Khusnutdinov et al. 2021). The Cas9 mutagenesis of TT8 (NtAN1a
and NtAN1b) in tobacco (Tian et al. 2021) and BnTT8 in Bassica napus
(Zhai et al. 2020) induced depigmentation of seeds and flowers. The
MYB TFs members of MBW have also been identified through CRISPR
analysis. The SlAN2 and R2R3-MYB TF, members of MBW in tomato,
have been reported through Cas9. The mutants accumulated much less
anthocyanin than wild-type (Yan et al. 2020). DcMYB113-like encoding
of the R2R3-MYB TF member of the MBW complex was revealed after
Cas9 editing in deep purple carrot, resulting in purple pigmentation, and
knockout of DcMYB7 in purple carrot resulted in yellow color (Xu et al.
2019). Besides, in some species-specific cases like maize, a combination
of MYB and bHLH transcription factors is not required (Khusnutdinov
et al. 2022). In fact, in the complex, the MYB proteins of the MBW
complex are thought to be the primary elements of the MBW complex
that activate the target genes because they bind to DNA. MYB has a
specialized function in regulating the production of anthocyanins.
Hence, variations in the intensity and pattern of the color are typically
explained by variations in the expression of the MYB TFs in the MBW
complex. The basic helix-loop-helix (bHLH) partners in the MBW com­
plex may have a variety of separate roles that extend beyond the control
of anthocyanin biosynthesis. The WD40 protein functions as a scaf­
folding molecule, supporting other proteins’ appropriate functions
(Mackon et al. 2021a; Xia et al. 2021). Numerous crops, including
purple-fleshed sweet potatoes, purple cauliflower, red-fleshed apples,
blood oranges, and purple pummelo, have high levels of anthocyanin
accumulation that are caused by elevated expression of MYB (Xu et al.
2015). Thus, MYB TFs are considered the main players in the complex
(Lloyd et al. 2017; Xu et al. 2015). Several studies have found that it can
either suppress or increase anthocyanin production in plants (Cappellini
et al. 2021; Khusnutdinov et al. 2022; Li et al. 2022a; Lloyd et al. 2017).
The MBW-complex function can be directly or indirectly altered by
repressors, which negatively affect the accumulation of anthocyanin (Li
et al. 2022a). In a direct way, the repressor either recruits some members
of the complex or blocks the binding site, preventing its formation. Kim
and co-workers reported BrMYBL2.1, an R3 MYB TF that suppressed
anthocyanin biosynthesis in cabbage by blocking MBW complex activity
(Kim et al. 2022). Khusnutdinov et al. reported two types of repressors,
AtMYB4-like and FaMYB1-like, that act by binding to the MYB motifs in
the promoters of structural genes and MBW complexes, respectively, and
replacing the positive MYB regulators (Khusnutdinov et al. 2021).
Further studies in apples revealed that MdMYB16 directly binds to the
5
Plant Science 333 (2023) 111746
promoters of MdANS and MdUFGT, preventing the formation of the
complex. In another study, PtrMYB57 blocked the formation of the
normal MBW anthocyanin activator complex by recruiting bHLH131
and PtrTTG1 (Wan et al. 2017). Likewise, other repressors may indi­
rectly control the complex activity by binding to the cis-element in the
gene’s promoter region to control the transcript levels of R2R3-MYB
activators, or likely through interactions with other upstream TFs or
by modulating their expression levels. Recently, Yang et al. highlighted
different TF family members from different signaling pathways,
including bZIP, WRKY, NAC, ERF/AP2, and HD-ZIP, that function as the
upstream regulators of the anthocyanin-related R2R3-MYBs, most of
which were identified in Arabidopsis and apple (Yang et al. 2022). Wang
and co-authors revealed a transcriptional repressor, AtGL2, in Arabi­
dopsis that negatively regulated anthocyanin biosynthesis by directly
repressing the expression of AtMYB113 and AtPAP2 MBW component
genes(Wang et al. 2015a). Similarly, MdBBX37, a B-BOX (BBX) protein,
was identified to negatively regulate anthocyanin biosynthesis by
interacting with two positive anthocyanin regulators MdMYB1 and
MdMYB9, inhibiting their binding to their target genes (An et al. 2019).
These numerous studies revealed that the regulation of anthocyanin is a
more complex process, which remains an important aspect of anthocy­
anin biosynthesis in plants.
3.3. Transporter’s genes
Anthocyanin in plants is synthesized on the external surface of the
endoplasmic reticulum (ER) and stored in the central vacuole at a high
concentration before the color is visible (Gomez et al. 2009; Landi et al.
2015; Mackon et al. 2021b; Poustka et al. 2007). Transporters were
involved in the trafficking mechanism from the site of biosynthesis to
the site of accumulation. Based on the model described by Grotewold
and Davies in 2008, the schematic representation by Gomez et al.
(2009), and the conceptual model proposed by Mackon et al. (2021a),
three main transporter families, including a glutathione-S-Transferase
(GST), an ATP binding cassette (ABC), and a multidrug and toxic com­
pound extrusion (MATE), were identified. Among these three families,
MATE and GST were the most important.
MATE has been implicated in the transport of various anthocyanin
types, such as acylated anthocyanin VvAM1 and VvAM3 from Vitis
vinifera (Gomez et al. 2011); flavone glycoside or malonylated antho­
cyanin MtMATE2 from Medicago truncatula (Zhao et al. 2011); and four
others were not specific, such as SlMATE or MTP77 from Solanum lyco­
persicum (Mathews et al. 2003), RsMATE8 from Rativus sativus (M’mbone
et al. 2018), and AtFFT from Arabidopsis thaliana (Thompson et al.
2009), OsMATE34 in Oryza sativa (Mackon et al. 2021c) associated with
the transport and accumulation of glycosylated anthocyanin inside the
vacuole.
GSTs are the most important family transporters. The first GST gene
implicated in anthocyanin accumulation in maize was Bronze-2 (Bz2)
(Marrs et al. 1995). Many plants, including Arabidopsis, have Bz2 ho­
mologous genes (TT19). GST were also identified in many crops such as
MdGST6 in apple (Jiang et al. 2019b), GSTU34 in rice (Ma et al. 2015),
DcGSTF2 in carnations (Sasaki et al. 2012), MrGST1 in Chinese bayberry
(Xue et al. 2022), CmGST1 in chrysanthemum (Li et al. 2021), GhTT9 in
cotton (Chai et al. 2023), LcGST4 in Litchi (Hu et al. 2016), MtGSTF7 in
Medicago truncatula (Wang et al. 2022a; Panara et al. 2022), LhGST in
Lilies (Cao et al. 2021), PpGST1 in peach (Zhao et al. 2020), AN9 in
petunia (Mueller et al. 2000), RsGSTF12 in radish (Niu et al. 2022). The
GST proteins for anthocyanin sequestration are highly conserved and
mainly bind to anthocyanin at the cytoplasmic surface of the endo­
plasmic reticulum and catalyze the formation of GSH-conjugated
anthocyanin; they may also increase the water solubility of anthocy­
anin to prevent its degradation (Niu et al. 2022).
Whether the above transporters worked separately or together and
their spatio-temporal activity in plants is still unclear, which could bring
more information about the storage mechanism of anthocyanin.
E. Mackon et al. Plant Science 333 (2023) 111746

(A) Schematic pathway of anthocyanin biosynthesis in plants; early
biosynthesis genes (EBG) are highlighted in red boxes, and late biosyn­
thesis genes (LBG) are highlighted in yellow boxes. (B) Regulatory process
of anthocyanin; the structural genes, decorating genes, and transporters
and mainly regulated by the conserved MYB-bHLH-WDR (MBW) com­
plex. (C) Storage mechanism of anthocyanin inside the cell; the complete
conceptual model of the storage mechanism can be found in our previous
review (Mackon et al. 2021a). Enzyme names are abbreviated as follows:
phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H),
4-coumarate:CoA ligase (4CL) chalcone synthase (CHS), chalcone isom­
erase (CHI), flavanone 3β-hydroxylase (F3H), flavonoid 3’-hydroxylase
(F3’ H), flavonoid 3’,5’ -hydroxylase (F3’5’H), dihydroflavonol 4-reduc­
tase (DFR), anthocyanidin synthase (ANS), leucoanthocyanidin dioxyge­
nase (LDOX), uridine diphosphate-dependent glucosyltransferase
(UGT/UFGT), glucosyltransferase (GT), acyltransferase (AT), methyl­
transferase (MT) rhamnosyltransferase (RT).
4. Application of CRISPR-Cas9 in anthocyanin improvement
Targeted genome editing is a quick, long-lasting, and affordable
method for enhancing anthocyanins in plants. Many genes have been
revealed to be important in anthocyanin pigmentation. However, the
knockout of these genes may result in color reduction. The use of
CRISPR/Cas9 to enhance anthocyanin involves initially finding key
relevance genes that, when expressed, inhibit or activate the anthocy­
anin process. Basically, the genes that cause phenotype diversity may be
found using both forward and reverse genetics methods (Lau et al.
2015). The primary source of diversity for conventional plant breeding
techniques is indeed the available germplasm. Following extensive
population screening, which takes a lot of time and effort, repeated
back-crossing is needed to introduce desired characteristics into the
chosen germplasm. However, using targeted genome alteration and
reverse genetic methods to speed up plant breeding and improve
pigmentation by knocking out a negative regulator of anthocyanin
production may appear simple. The information that is now accessible
on CRISPR/Cas9 has encouraged its use in the genetic enhancement of
anthocyanin in plants (Table 3).
D’Amelia and colleagues recently increased and stabilized anthocy­
anin production in potato cell cultures of variety "Blue Star" by using
CRISPR/Cas9 to knock out a gene called Inducer Silencing of Anthocy­
anins in Cell Cultures (StlSAC). StlSAC is an R3-MYB transcription factor
previously named StMYBATV (D’Amelia et al. 2022), which repressed
the formation of the MYB-bHLH-WD40 by recruiting the bHLH
co-partner of the MBW complex (D’Amelia et al. 2022). Phylogenetic
analysis revealed relatedness to SlMYBATV, an anthocyanin repressor in
tomato (Cao et al. 2017). The knock-out of StlSAC led to the deletions
that caused a frame-shift mutation and a loss of function, resulting in a
dark purple color in contrast to the mosaic white color in the WT. The
edited cells produce about threefold more anthocyanin compared to WT
cells and 14-fold more compared to potato tubers (Table 3).
The FtMYB45 gene is an R2R3-MYB transcriptional repressor of
flavonoid biosynthesis found in Tartary buckwheat (Fagopyrum tatar­
icum), a valuable edible and medicinal crop (Zhang et al. 2018). A
polycistronic tRNA-SgRNA-mediated CRISPR-Cas9 has been applied to
knock out the FtMYB45, resulting in an increased amount of eight fla­
vonoids, including proanthocyanidin and anthocyanin content.
FtMYB45 acts by binding directly to FtPAL, affecting the entire flavonoid
biosynthesis pathway (Wen et al. 2022).
In Malus domestica, the CRISPR mutagenesis analysis revealed that
MdMYB16 inhibits the expression of the LBG genes MdANS and MdUFGT
by direct binding to their promoters and reduces anthocyanin accumu­
lation in apple calli (Xu et al., 2017). The knockout form of MdMYB16
doubles the amount of anthocyanin. MdMYB16 also interacted with
MdbHLH33, is responsible for the formation of homodimers, and, via its
C-terminal EAR repressor motif, directly suppresses anthocyanin pro­
duction. Most R2R3-MYB TFs possess, in addition to a DNA binding
domain, an ethylene-responsive element binding factor-associated
amphiphilic repression (EAR) or the arrangement of TLLLFR amino
acid sequence, an active transcriptional repression motifs at the C ter­
minus, which exerts transcriptional control over genes involved in the
production of structural anthocyanins as well as transcription factor
genes.
The binary pYLCRISPR/Cas9 multiplex construct carrying three
SgRNAs has been successfully applied to knock-out PtrMYB57, an R2R3-
MYB anthocyanin repressor TF in poplar. The mutants generated a two-
fold higher level of anthocyanin content compared to the wild-type.
Further investigations revealed that the expression level of structural
genes PAL4, 4CL5, CHS, CHI, F3H, DFR1, ANS1, ANR1, and LAR1 was
increased in the mutant lines. PtrMYB57 blocked the formation of the
normal MBW anthocyanin activator complex which controls the
expression level of the above structural genes, by recruiting bHLH131
and PtrTTG1 (Wan et al. 2017). A similar effect was obtained by
over-expressing the second repressor, PtrMYB182, in hairy roots and
transgenic plants of poplar. The PtrMYB182 repressed MYB134, a

Table 3
Previous studies on CRISPR/Cas9-mediated anthocyanin enhancement.
Plants Gene Function Targets Methods Editing events Reference

Potato StlSAC R3-MYB MBW complex CRISPR/CAS9 Knock-out The edited cells produce approximately three times as much (D’Amelia et al.
TF anthocyanin as WT cells and fourteen times as much as 2022)
potato tubers.
Tartary FtMYB45 R2R3 FtPAL polycistronic tRNA- Whole flavonoid biosynthesis pathways and increased (Wen et al.
buckwheat MYB TF SgRNA-mediated CRISPR- anthocyanin 2022)
Cas9
Apple MdMYB16 R2R3- MdANS and the CRISPR mutagenesis The knockout of MdMYB16 doubles the amount of (Xu et al. 2017)
MYB TF MdUFGT knock-out anthocyanin.
Poplar PtrMYB57 R2R3- PtrbHLH131 and The binary pYLCRISPR/ The mutants generated a 2-fold higher level of anthocyanin (Wan et al.
MYB TF PtrTTG1 Cas9 multiplex content compared to the wild type. 2017)
Poplar PtrMYB182 R2R3- PtrMYB134 CRISPR-CAS9 Knock-out The mutants generated a 2-fold higher level of anthocyanin (Yoshida et al.
MYB TF content compared to the wild type. 2015)
Rapeseed BnMYB60 R3-MYB MBW complex Crispr-knock-out (Mikhaylova
TF et al. 2020)
Arabidopsis AtMYBL2 R3-MYB AtDFR and AtTT8 RNAi Knock-out Ectopic accumulation of anthocyanin in Arabidopsis leaves (Matsui et al.
TF TF 2008)
Tomato SlMYBATV R2R3- MBW complex CRISPR/Cas9-mediated The amount of anthocyanin in the mutant Slmybatv was 12- (Sun et al. 2020)
MYB TF knock-out fold higher than in the wild type SlAN2-like/Aft and
comparable to indigo red cv.
Tomato SlANT1 R2R3- MBW complex CRISPR knock-in Ectopic accumulation of pigments in tomato tissue (Čermák et al.
MYB 2015)
Arabidopsis PAP1 R2R3 MBW complex CRISPR/dCas9 activator Pap1dCas9-D transgenic plants， with a three - fold increase (Park et al.
MYB TF system in PAP1 expression had a brilliant purple hue 2017)

6
E. Mackon et al.
positive regulator of transcriptional activator of anthocyanin leading to
low level of anthocyanin (Yoshida et al. 2015). The orthologs of
AtMYB60, AtCPC, and AtMYBL2 in Brassica napus L (oilseed, rapeseed)
were edited through CRISPR/Cas9, and the result revealed an increasing
anthocyanin in the leaves. However, BnMYB60 showed a more efficient
result. In addition, the mutants were also stress-tolerant. The study
provided the possibility to increase anthocyanin pigmentation in
B. napus through Cas9-mediated knock-out of three TFs: MYB60, CPC,
and MYBL2, all inhibitors of the MBW complex (Elena et al. 2022).
Another study showed that the R3-MYB protein of AtMYBL2 functions as
a transcriptional repressor and inhibits the production of anthocyanins.
AtMYBL2 knock-out lines enhanced the expression of DFR and TT8 TF,
resulting in the ectopic accumulation of anthocyanin in Arabidopsis
leaves (Matsui et al. 2008). These results indicated that AtMYBL2 and
PtrMYB182 are potential candidates for increased anthocyanin by
CRISPR/Cas9 knock-out. Myriad anthocyanin repressors similar to these
have been deciphered, showing a promising future for
CRISPR-Cas9-mediated anthocyanin accumulation in plants.
In tomato, SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft are the
four anthocyanin-related R2R3-MYB TFs (Zhi et al. 2020). Functional
SlAN2-like can activate the expression of both anthocyanin biosynthetic
genes and their regulatory genes. It was shown that cultivated tomatoes
contain nonfunctional alleles of SlAN2-like and therefore fail to produce
anthocyanin. Indigo-rose cv. tomatoes with purple color have a domi­
nant SlAN2-like gene or Aft with a recessive SlMYBATV, but anthocyanin
accumulation in the fruit skin is light-dependent. SlMYBATV encodes a
negative regulator of anthocyanin biosynthesis. In general, SlMYBATV
and SlAN2-like molecules compete for binding to SlAN1. Sun et al.
(2020), achieved high anthocyanin production in tomatoes via
Cas9-mediated knocked-out of SlMYBATV. The amount of anthocyanin
in the mutant Slmybatv was 12-fold higher than in the wild type
SlAN2-like/Aft and comparable to indigo red cv. The overexpression of
SlMYBATV leads to very green-colored tomatoes. Consistently, expres­
sion of a functional SlAN2-like gene driven by the fruit-specific promoter
in a tomato cultivar led to the activation of the entire anthocyanin
biosynthesis pathway and high-level accumulation of anthocyanins in
both the peel and flesh. Additionally, a CRISPR knock-in approach was
successfully used to insert the constitutive CaMV-35S strong promoter
upstream of the SlANT1 anthocyanin gene pigmentation, resulting in the
overexpression and ectopic accumulation of pigments in tomato tissue.
The bean yellow dwarf virus (BeYDV) was used as the vector to deliver
the donor template, gRNA, and Cas9 cassette to target SlANT1 through
Agrobacterium-mediated transformation, which produced dark purple
plants (Čermák et al. 2015). Two guide RNAs and Cas12a nuclease were
used to successfully replicate the experiment (Vu et al. 2020). This result
showed that CRISPR-Cas GET can be used specifically to knock-in gene
and promoter for great achievement in anthocyanin accumulation.
Cas9 has gone through a series of functional changes, from active
endonuclease to partially or completely inactive Cas9. With the inclu­
sion of activator or repressor domains, the catalytically deactivated Cas9
(dCas9) provides a platform for regulating transcriptional expression.
Researchers successfully utilized CRISPR/dCas9 to increase transcrip­
tional expression in plant cells. Piatek et al., generated a transcriptional
activator and repressor by fusing the dcas9 C-terminus with the activator
domain of EDLL and TAL effectors and the dCas9-terminus with the
SRDX repression domain, respectively (Lowder et al. 2015; Piatek et al.
2015). Similar to previous authors, Park and co-authors completely
redesigned CRISPR-Cas9 with a transcriptional activator to target pro­
duction of anthocyanin pigment (PAP1) and induce purple anthocyanin
in Arabidopsis (Park et al. 2008). PAP1 is an MYB TF whose over­
expression results in purple-colored Arabidopsis and tobacco due to the
accumulation of anthocyanin (Borevitz et al. 2000). The expression of
the PAP1 gene was evaluated under intense light. Identical to PAP1
over-expressors, Pap1dCas9-D transgenic plants with a three-fold in­
crease in PAP1 expression had a brilliant purple hue (Park et al. 2017).
Hence, the modified CRISPR/Cas9 activation system with p65-HSF
7
Plant Science 333 (2023) 111746
activators offers a straightforward method for creating activated plants
by enhancing endogenous transcriptional levels. These findings repre­
sent a remarkable achievement for anthocyanin enhancement.
5. CRISPR/Cas 9 perspectives toward anthocyanin enhancement
Cas9 has gone through a series of functional changes, from active
endonuclease to partially or completely inactive Cas9. Today, there are
flourishing opportunities for using CRISPR-Cas9 to enhance anthocy­
anin in plants.
5.1. CRISPR opportunities to improve anthocyanin
To date, more than 50 plant species (~100 genes) have been suc­
cessfully edited using the CRISPR/Cas9 system (Hajiahmadi et al. 2019;
Wang et al. 2022b; LaFountain and Yuan, 2021). However, they are
mostly used to identify gene function. Enhancing anthocyanin in plants
could be achieved through the knock-out of repressor genes or the
knock-in of the activator, which give huge opportunities for using
CRISPR-GET.
Myriad repressors or activators have not been explored yet by
CRISPR-Cas9. However, because knocking out repressor genes was the
first goal of CRISPR-technology, which has high efficiency, is relatively
simple, and is widely used, we focused on them here. In the recent
decade, several anthocyanin repressors have been discovered. Most of
these repressors either destabilize the MBW complex via protein-protein
interactions or suppress the transcription of MBW complex components
(LaFountain and Yuan, 2021). Among them, MYB TF is the most
promising CRISPR/Cas9 target, which was reported to inhibit anthocy­
anin production in Arabidopsis, poplar, apple, and other plants
(Table 4). Some are R2R3-MYB TFs, although most are R3-MYB TFs.
Overexpression, RNAi, CRISPR/Cas9, and VIGS are used to study plant
MYB TFs (Li et al. 2022a). Arabidopsis genes AtMYB60, AtCPC, and
AtMYBL2 were identified as negative regulators with an R3 motif. Their
overexpression repressed the expression of structural genes and
decreased anthocyanin synthesis and pigmentation (Zhu et al. 2009;
Matsui et al. 2008). The overexpression of PtrMYB182 in poplar resulted
in a decreasing anthocyanin level, suggesting that PtrMYB182 was a
negative regulator of anthocyanin biosynthesis through the MBW com­
plex (Yoshida et al. 2015). Similarly, the overexpression of PhMYBx1 in
petunia (Fu et al. 2019), FtMYB8 in tartary buckwheat (Huang et al.
2019b), and MdMYBL2 in apple (Wang et al. 2019), decreased the
production of anthocyanin in flower, leaves, and fruit, respectively.
These genes are prominent targets for increased anthocyanin. In addi­
tion, Xiang et and co-workers showed that CmMYB#7, an R3-MYB TF,
inhibited anthocyanin production in chrysanthemum (Xiang et al.
2019). LhR3MYB1 and LhR3MYB2 harbored a C2 suppressor motif
downstream of a single MYB repeat, inhibiting lily anthocyanin
biosynthesis passively (Sakai et al. 2019); whereas, LvMYB1, an
R2R3-MYB transcription factor, suppressed lily flower anthocyanin
production (Yin et al. 2021). an R2R3-MYB TF, VvMYBC2L2, from
rose-repressed grapevine anthocyanin production (Zhu et al. 2018).
Deng and co-authors, identified MaMYB4 as an R2R3-MYB tran­
scription repressor in banana. MaMYB4 was characterized by EAR and
TLLLFR motifs. The overexpression of MaMYB4 results in the decreasing
of anthocyanin (cyanidin and delphinidin). Further investigations
showed that MaMYB4 blocked the promoters of CHS, ANS, and DFR and
interfered with the proper MBW complex to activate their expression
(Deng et al. 2021). The FaMYB1 gene was revealed as a potential tran­
scriptional repressor in strawberries. RNAi-mediated silencing has been
employed to identify FaMYB1 in strawberries (Aharoni et al. 2001) and
PhMYB27 in petunias. These genes suppressed ANS, GT, and DFR, which
are important in anthocyanin accumulation. The overexpression of
FaMYB1 and PhMYB27 significantly reduced the amount of anthocyanin
in all tissues. In many other species, MYB2 functions as a positive
regulator. However, MtMYB2, found in Medicago truncatula, repressed
E. Mackon et al. Plant Science 333 (2023) 111746

Table 4
Promising Crispr/Cas9-targeting for anthocyanin enhancement.
Plants Repressor genes Target of repressors Detection methods Reference

Arabidopsis AtMYBL2 DFR, LDOX, GL3, TT8, and PAP1 Overexpression (Matsui et al. 2008)
Arabidopsis AtCPC DFR, LDOX, CHS, CHI, F3’H, and F3H Overexpression (Zhu et al. 2009)
Poplar AtMYB182 MBW complex Overexpression (Yoshida et al. 2015)
Petunia AtMYBx1 MBW complex Overexpression (Fu et al. 2019)
Tartary buckwheat FtMYB8 TT12 Overexpression (Huang et al. 2019b)
Apple MdMYBL2 MdDFR and MdUFGT, MdMYB10 and Overexpression (Wang et al. 2019)
MdbHLH3 TF
Chrysanthemum CmMYB#7 MBW complex Transient- overexpression (Xiang et al. 2019)
Lily LhR3MYB1 MBW complex Stable and transient- overexpressed (Sakai et al. 2019)
LhR3MYB2
Lily LvMYB1 ANS Transient- expression (Yin et al. 2021)
Grapevine VvMYBC2L2 CHS, DFR, LAR and UFGT Overexpression (Zhu et al. 2019)
Tobacco GtMYB1R1 MBW complex Overexpression (Nakatsuka et al. 2013)
GtMYB1R9
Tobacco AtCPC MBW complex Overexpression (Zhang et al. 2009)
Gerbera GhMYB1a CHS, F3H, and FLS. Overexpression (Zhong et al. 2020)
Grape hyacinth MaMYBx MabHLH1 Overexpression (Zhang et al. 2020)
Chinese cabbage BrMYBL2.1 MBW complex Transient- promoter activation assay (Kim et al. 2022)
Apple MdMYB16 ufgt and DFR promoter Overexpression (Xu et al. 2017)
Banana MaMYB4 CHS, ANS, DFR, and bHLH Overexpression and metabolome (Deng et al. 2021)
analysis
Arabidopsis AtGL2 MBW complex Transient- expression (Wang et al. 2015b)
Strawberry FaMYB1 ANS, GT and DFR RNAi-mediated silencing and (Aharoni et al. 2001; Kadomura-Ishikawa et al.
overexpression 2015);
Petunia PhMYB27 ANS, GT and DFR Overexpression (Albert et al. 2014)
Medicago MtMYB2 UFGT Overexpression (Jun et al. 2015)
truncatula
Arabidopsis HAT1 DFR, LDOX and UF3GT Overexpression (Zheng et al. 2019b)
Brassica oleiracea NAC019 PAL, C4H, CHS, F3H, ANS and UFGT Overexpression (Wang et al. 2018)
Litchi LcNAC13 CHS, CHI, F3H, F3’H, DFR, and MYB1 Transient expression (Jiang et al. 2019a)
Arabidopsis MiR858a MYBL2 Generation of (Wang et al. 2016)
transgenic plants,
expression profiling
Potato miR858 MYB12 Generation of (Lin et al. 2021)
transgenic plants
kiwifruit miR858 MBW complex Overexpression (Li et al. 2020b)
Arabidopsis AtMYB60 DFR Overexpression (Park et al. 2008)
Tartary buckwheat FtMYB1 and FtMYB2 MBW complex Overexpression (Bai et al. 2014)

anthocyanin accumulation. The overexpression of MtMYB2 led to a
decrease in the amount of anthocyanin, while mutants had increased
anthocyanin. Further analysis showed that it may principally target the
UFGT structural gene (Jun et al. 2015). Other key genes for improving
anthocyanin have been revealed. Among these, R3-MYB TF BoMYBL2–1
in Brassica oleracea, IlMYBL1 in Iochroma, PhMYBx in Petunia, PtrRML1
in Poplar, ROI1 in Rose, and GtMYB1R1 and GtMYB1R9 in Gentiana
triflora have been extensively studied (Chen et al. 2019). They nega­
tively regulate anthocyanin through the inhibition of the MBW complex
(Table 4).
Other studies discovered non-MYB anthocyanin repressors, some of
which belonged to the Lateral Organ Boundary Domain (LOBD) TF
family; NAC, HAT1, and miRNA are underappreciated as CRISPR/Cas9
targets. The MdLBD13 from apple was overexpressed in Arabidopsis
resulting in the decreasing anthocyanin accumulation (Li et al. 2017a).
In Pyrus bretschneideri (pear), the PbrLBD20, PbrLBD35, and PbrLBD53
genes showed increased pigmentation when down-regulated (Song et al.
2020). In B. oleracea, BoLBD39 and BoLBD37 were associated with
anthocyanin degradation (Ren et al. 2019). Similarly, the over­
expression of homeobox Arabidopsis thaliana 1 (HAT1) reduced
anthocyanin pigmentation. HAT1 belongs to the HD-ZIP family with an
N-terminal EAR motif. HAT1 interacted with MYB75, inhibiting the
formation of the MBW complex, which repressed the expression of the
DFR, LDOX, and UF3GT genes (Zheng et al. 2019b). HAT1 knock-out of
the EAR motif increases anthocyanin content. Members of the NAC were
also identified. Overexpression of NAC019 in Brassica oleracea repressed
PAL, C4H, CHS, F3H, ANS, and UFGT genes and decreased anthocyanin
accumulation (Wang et al. 2018). The transient expression of LcNAC13
litchi in tobacco downregulated the expression of CHS, CHI, F3H, F3’H,
DFR, and MYB1 by directly binding to their NACs motif and repressing
anthocyanin accumulation (Jiang et al. 2019a). Micro-RNAs influence
gene expression by cleaving target mRNAs or inhibiting gene trans­
lation. In A. thaliana, miR858 downregulated MYB11, MYB12, and
MYB111, which directly induced early biosynthesis genes. MiR858a
inhibited MYBL2 to increase anthocyanin (Wang et al. 2016). MYB12
gene repression by miR858 lowered potato’s flavonol content (Lin et al.
2021). In kiwifruit, miR858 overexpression blocked anthocyanin pro­
duction (Li et al. 2020b).
In most recent studies, researchers mainly focused on coding regions
to produce loss-of-function mutants and enhance anthocyanin. Howev­
er, targeting cis-regulatory motifs, also called cis-engineering, is very
important in fine-tuning gene expression and generating phenotypic
variation (Rodríguez-Leal et al. 2017). With the advent of CRISPR/Cas9,
cis engineering has become a more sophisticated approach to generating
crop diversity and improving agronomic traits. Compared to coding
sequences, it may produce less detrimental deleterious effects and offer
alternative mutagenesis possibilities, as it is used for promoter disrup­
tion, insertion, swapping, and cis regulatory element (CRE) disruption
and deletion (Li et al. 2020a). Several studies reported the use of the
CRISPR/Cas-mediated cis regulatory region in different crops (Li et al.
2020a; Jia et al. 2016; Li et al. 2017b; Shrestha et al. 2018). In a recent
study, Li and co-workers used CRISPR/Cas9 to generate allelic variation
in the tomato KLUH promoter, gene controlling organ size ( reverse of
HULK a comic monster)(Anastasiou et al. 2007), by targeting a single
nucleotide polymorphism highly associated with a conserved CRE. As a
result, the fruit weight significantly increased, while the proportion of
small fruit decreased (Li et al. 2022b). Liu et al. edited the promoter of
the CLAVATA3/ESR-RELATED (CLE) genes, a small polypeptide family

8
E. Mackon et al.
sharing several structural features and a null allele of the CLE gene in
maize, which resulted in the change of yield-related traits and yield
improvement (Liu et al. 2021b). Likewise, novel Waxy alleles with
fine-tuned amylose content and improved rice grain quality were
generated through CRISPR/Cas9 after editing the region near the TATA
box cis-element of the Wxb promoter (Huang et al. 2020). The use of
CRISPR/Cas-mediated cis-regulatory regions to enhance anthocyanin is
still less explored, but it remains one of the most practical strategies to
improve the amount of anthocyanin in plants without affecting the
genes or coding sequences.
5.2. Limitations of CRISPR-Cas approach in improving anthocyanin
content
The CRISPR/Cas9 system brought breakthrough progress to the
research and application of targeted genome modification, especially in
the fields of gene function verification, disease-targeted therapy, and
crop improvement. But there are still many bottlenecks and challenges
in the application (Fig. 3).
5.2.1. Off-target mutations
Off-target (unwanted) mutations are a significant issue in genome
editing because they may bring about undesirable changes in plants
when they happen in a coding region. Off-targets can cause chromo­
somal rearrangements, harming incompletely matched genomic regions
and limiting GE’s therapeutic potential. Off-target impacts may poten­
tially reduce gene activity, causing various physiological or signaling
issues (Manghwar et al. 2020). Most of the mutations occur during
transformation and in-vitro cultures. Although it is a serious threat in
animals, in plants it is uncommon and may be eliminated through
backcrossing. It was reported that only one of the twelve Cas9 sgRNAs
produced off-target changes (Graham et al. 2020), and no off-target
mutations were found after editing with Cas12a (Khusnutdinov et al.
2021). Nonetheless, reducing off-target effects in plants has become a
Fig. 3. CRISPR/Cas9 limitations for anthocyanin improvement.
9
Plant Science 333 (2023) 111746
research challenge, and several approaches have been proposed,
including dcas9 and cas9 paired nickase (Hajiahmadi et al. 2019; Hahn
and Nekrasov, 2019). These could help improve CRISPR-Cas9 GET
efficiency.
5.2.2. Gene flow and Cas’s activity persistence
CRISPR-Cas-edited crops are also hindered by gene flow. Genetic
migration of altered gene sequences or off-target mutations from
CRISPR-Cas-edited organisms to wild-type species may have environ­
mental effects. A CRISPR-Cas’s gene modified to confer drought toler­
ance in rice, for example, could evolve into a noxious weed. Cas’s
activity in future generations is another CRISPR-Cas’s concern. The re­
sidual presence of Cas could result in a potentially harmful mutation in a
genetically stable lineage. In an Arabidopsis investigation, it was
discovered that Cas activity persisted through successive generations
(T3) (Feng et al. 2016).
5.2.3. Public concerns and safety
Despite being a strong and accurate technique for genome editing in
many organisms, CRISPR/Cas9 systems are still not frequently used,
which can be explained by public concerns about their safety. In fact,
CRISPR-editing requires the stable insertion of T-DNA (containing Cas,
gRNAs, and selection markers), which is considered foreign DNA in the
plant genome (Ma et al. 2015; Lowder et al. 2015). Therefore, they are
also regarded as genetically modified organisms (GMOs). As such, in the
EU, Australia, and New Zealand, transgenic laws are applied to all edited
plants, including all CRISPR types, whereas in the US and Canada, the
transgenic regulatory process is applied only to plants with foreign DNA
(Menz et al. 2020; Ahmad et al. 2021). However, CRISPR plants remain
more environmentally friendly and sustainable (Khusnutdinov et al.
2021; Menz et al. 2020). Nonetheless CRISPR-Cas should be repurposed
and clean gene edited plants will be of great interest (Vu et al. 2020). So,
it is important to develop efficient methods that do not allow foreign
DNA in plants. Recent attempts have been made to overcome this issue.
E. Mackon et al.
Recently, He et al. used a visible marker, a unit that activates anthocy­
anin biosynthesis coupling with the CRISPR/Cas9 cassette, that was
called the anthocyanin-marker assisted CRISPR (AAC) technology, to
identify transgene-free plants at the callus stage and in the field (He et al.
2020). Likewise, Gao and co-authors successfully isolated an heritable
and cas9-free mutant using a mCherry fluorescence marker as a proxy
for the presence of the CRISPR/Cas9construct (Gao et al. 2016a).
Another study employed a pair of suicide transgenes that naturally
eliminated all pollen and embryos containing CRISPR/Cas9 in T0 and T1
plants, reducing labour and time and avoiding the spread of the trans­
gene plants in nature (He et al. 2018). In a similar study, an RNA
interference element targeting an herbicide-resistance P450 enzyme was
associated with the CRISPR construct to isolate a transgene-free mutant
(Lu et al. 2017). Vu and co-workers used viruses like Bean yellow dwarf
virus (BeYDV), which initiate rolling circle replication of CRISPR ele­
ments inside T-DNA without integration into the genome. There are still
many promising model genes that have not been used as CRISPR targets
yet （Table4）. However, as long as all edited plants are subjected to the
transgenic process, this could be a great limitation of CRISPR in
anthocyanin improvement.
6. Conclusion
Anthocyanin is one of the most important plant secondary metabo­
lites with tremendous advantages, including alleviating biotic and
abiotic stresses, use as a natural food additive, use as a visual marker,
and health promotion. The latter function is expected to be performed
efficiently while consuming a large amount of anthocyanin. Anthocy­
anin biosynthesis has been widely studied, and structural and regulatory
genes are now elucidated. Recently, anthocyanin activators and re­
pressors have been deciphered, with MYBTFs as the most represented.
These conditions may favor future investigations toward anthocyanin
enhancement.
Today, the CRISPR/Cas9 system has all the potentialities of genome
editing, such as knock-in, knock-out, knockdown, expression activation,
base editing, epigenome engineering, imaging, nuclear rearrangements,
etc., to achieve high anthocyanin in plants. However, most previous
studies focused on repressor MYBTFs in order to improve anthocyanin
through CRISPR/Cas9-mediated knock-out. This suggested that tran­
scription factors, mainly MYB TF families, play a crucial role in antho­
cyanin accumulation. Nonetheless, multiple approaches, such as knock-
in, expression activation through the CRISPR/Cas9 system, and other
promising targets, including microRNAs, structural genes, antagonist
enzymes (FNS, FLS, and IFS), and other proteins (the MADS, NAC, HD-
Zip, and ERF TF families), which are also implicated in anthocyanin
synthesis, need to be investigated. This will offer huge possibilities to
improve anthocyanin through the CRISPR/Cas9 system and enable the
creation of novel biotechnological methods to produce value-added
plants with higher anthocyanin content, helping consumers get the
right quantity in their diet. Exploring genes involved in anthocyanin
modification, such as AT and GT, can help to achieve the desirable
quality of anthocyanin and manipulate color shade, which is important
for flower producers.
Although CRISPR-Cas technology has recently been revealed as a
favorite researcher’s tool, significant drawbacks have been encountered,
including time consumption, the introduction of foreign DNA, off-target
issues, and, the most tricky of all, regulation and bioethics. Thus, it is
important to repurpose and create more precise and organized ap­
proaches such as unbiased and biased off-target detection techniques,
gRNA engineering and modification, enhanced Cas’s variants, effective
CRISPR system delivery techniques, anti-CRISPR protein development,
and efficient base-editing and prime editing systems. This could facili­
tate the development of risk-free gene editing crops, which may increase
their acceptability.
10
Plant Science 333 (2023) 111746
Funding
This work and APC were funded by the Guangxi Provincial R&D
Priority Program for innovation-driven Development-Breeding and
Demonstration of Elite New and High-Quality Rice Varieties with Low
Cadmium and Resistance, Guike AA22068087.
CRediT authorship contribution statement
Enerand Mackon and Piqing Liu: Conceived the project. Enerand
Mackon and Guibeline Charlie Jeazet Dongho Epse Mackon: Drafted
the manuscript. Yongqiang Guo, Yafei Ma, and Yuhang Yao: Edited
table, graphs and revised the manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest.
Data Availability
No data was used for the research described in the article.
Acknowledgments
We are thankful to Izhar Ali for proofreading.
Institutional review board statement
Not applicable.
Informed Consent Statement
Not applicable.
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