cells

Perspective
Analysis of the Utilization and Prospects of CRISPR-Cas
Technology in the Annotation of Gene Function and Creation
New Germplasm in Maize Based on Patent Data
Youhua Wang 1 , Qiaoling Tang 1 , Yuli Kang 1 , Xujing Wang 1 , Haiwen Zhang 1, * and Xinhai Li 2, *

1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2 Institute of Crop Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement,
Chinese Academy of Agricultural Sciences, Beijing 100081, China
* Correspondence: [email protected] (H.Z.); [email protected] (X.L.)

Abstract: Maize (Zea mays L.) is a food crop with the largest planting area and the highest yield
in the world, and it plays a vital role in ensuring global food security. Conventional breeding
methods are costly, time-consuming, and ineffective in maize breeding. In recent years, CRISPR-Cas
editing technology has been used to quickly generate new varieties with high yield and improved
grain quality and stress resistance by precisely modifying key genes involved in specific traits, thus
becoming a new engine for promoting crop breeding and the competitiveness of seed industries.
Using CRISPR-Cas, a range of new maize materials with high yield, improved grain quality, ideal
plant type and flowering period, male sterility, and stress resistance have been created. Moreover,
many patents have been filed worldwide, reflecting the huge practical application prospects and
commercial value. Based on the existing patent data, we analyzed the development process, current
Citation: Wang, Y.; Tang, Q.; Kang, Y.;
status, and prospects of CRISPR-Cas technology in dissecting gene function and creating new
Wang, X.; Zhang, H.; Li, X. Analysis germplasm in maize, providing information for future basic research and commercial production.
of the Utilization and Prospects of
CRISPR-Cas Technology in the Keywords: maize; CRISPR-Cas technology; germplasm; patent data
Annotation of Gene Function and
Creation New Germplasm in Maize
Based on Patent Data. Cells 2022, 11,
3471. https://doi.org/10.3390/ 1. Introduction
cells11213471
Improvement of the yield, quality, and stress resistance of crops is the goal of scientists
Academic Editors: Yan Wenhao and and breeders. Conventional breeding, including cross-breeding and mutation breeding, has
Shuangxia Jin significantly improved crop yield and crop performance under various stresses over the last
century, but traditional breeding is limited by bottleneck factors such as a lack of available
Received: 28 September 2022
germplasm resources, unfocused breeding objectives, difficult pyramiding of elite traits, a
Accepted: 31 October 2022
long breeding process, and high cost. Although transgenic technology is undoubtedly an
Published: 2 November 2022
alternative to improve crops with desirable traits, its major limitation is the strict safety
Publisher’s Note: MDPI stays neutral regulatory procedures and the low public acceptance of genetically modified crops [1].
with regard to jurisdictional claims in Genetic variation and diverse germplasm are the basis of crop breeding. Gene-editing
published maps and institutional affil- technology can overcome the bottleneck of lack of germplasm resources in traditional
iations.
breeding by introducing precise genetic alterations to the targeted genomes, efficiently
accelerating the pyramiding of multiple traits [2]. Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)/CRISPR-associated proteins (CRISPR-Cas) technology
has become a powerful tool for crop genetic improvement by efficiently and accurately
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
modifying target genes, thereby accelerating plant breeding. In particular, different precise
This article is an open access article
gene-editing tools such as base editing, prime editing, and gene targeting provide tools
distributed under the terms and
for the precise aggregation of multiple traits in an elite variety. CRISPR-Cas technology
conditions of the Creative Commons has been used widely in the analysis of key genes and the creation of new germplasm in
Attribution (CC BY) license (https:// rice, maize, soybean, and wheat, thus starting a new era of molecular design breeding
creativecommons.org/licenses/by/ in crops [3–5]. In the United States, gene-edited crops may take only 5 years from initial
4.0/). research to commercial production. Gene-edited products such as waxy corn, high oleic

Cells 2022, 11, 3471. https://doi.org/10.3390/cells11213471 https://www.mdpi.com/journal/cells

Cells 2022, 11, 3471 2 of 12

acid soybean, and high-GABA tomato are being produced commercially in the US, Japan,
and other countries [6]. In 2022, the Guidelines for the Safety Evaluation of Gene Editing
Plants for Agricultural Use (Trial) was issued by the Ministry of Agriculture and Rural
Affairs of the People’s Republic of China, providing laws and regulations for the process of
creating, safety evaluating, and commercially producing gene-edited crops.
Maize (Zea mays L.) is a food crop with the largest planting area and the highest yield
in the world, and it plays a vital role in ensuring food security and alleviating energy
crises. Due to limited elite germplasm resources and cross-incompatibility, conventional
breeding methods are relatively insufficient and time-consuming, taking years to obtain
ideal materials, and biotechnologies have proven to be attractive in the creation of excellent
maize germplasm [7,8]. In particular, CRISPR-Cas can precisely and directly generate new
lines with multi-trait aggregation within 2 years by modifying genes controlling different
target traits [7]. Patenting is one of the most important indicators of technological innova-
tion and new material creation, and it is an important foothold in market competition. In
recent years, the research and applications of CRISPR-Cas gene-edited maize have become
increasingly competitive, and increasingly related patents have been filed worldwide. The
patent layout reflects the commercial prospects of gene-edited maize germplasm, indicating
the importance of intellectual property protection in seizing technological innovation and
market competitiveness in the future. This paper analyzes the research process, current
status, and development trends in global gene-edited maize from the perspective of the
relevant patent protections, which provides information for the macroplanning of future
research and the commercialization of gene-edited maize materials.

2. Rapid Advances in Using CRISPR-Cas Editing in Maize

We first analyzed the relevant patent applications for CRISPR-Cas technology in maize
(as of March 2022) using the global patent database search (https://analytics.zhihuiya.
com/, accessed on 29 May 2022). We found 184 patents (Column A of Supplementary Table S1)
on the use of CRISPR-Cas technology in analyzing gene function and the generation of new
germplasm in maize worldwide. Considering the layout of the same patent content in dif-
ferent countries and regions as one patent, 123 different patents were obtained (Column B
of Supplementary Table S1). Based on the number of patents per year, we identified an
exploration period transitioning to a rapid development period. From 2015 to 2018, there
were fewer than 10 patents per year (exploration period) (Figure 1). Since 2019, the related
research has entered a rapid development period with more than 20 patents filed every
year. Specifically, 48 patents were filed in 2021, accounting for 40.33% of the total patents
(Figure 1). Of the 123 patents, 104 were filed by Chinese scientific research institutes,
accounting for 84.55% of the total. In particular, great breakthroughs have been made in
developing new maize germplasm and innovative cross-breeding methods using CRISPR-
Cas technology (Supplementary Table S1). For example, the ZmRAVL1 maize gene and
functional site, and the use thereof, was filed by China Agricultural University, providing
a method of creating new maize germplasm with compact plant types suitable for dense
planting and high yield, accomplished by editing the ZmRAVL1 gene [9]. Another example
is the Artificial Creation of Maize Male Sterile Lines and Efficient Transformation Meth-
ods, created by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences,
which present a new way of creating male sterile lines and the utilization of heterosis in
maize [10]. These achievements indicate that the research and applications of gene-edited
maize in China are at the forefront of the world. Moreover, waxy corn hybrids with higher
yield were generated with CRISPR–Cas9 by editing waxy1 in 12 elite inbred maize lines.
Importantly, these CRISPR–Cas9 waxy corn hybrids were not subjected to the regulatory
oversight regarding genetically modified organisms in the United States, Argentina, Brazil,
and Chile. A limited precommercial launch was conducted in the Midwestern United
States in 2019 [11]. These major advances in CRISPR-Cas technology in maize have opened
up a new era for the generation of new germplasm and elite varieties.
 were not subjected to the regulatory oversight regarding genetically modified organisms
in the United States, Argentina, Brazil, and Chile. A limited precommercial launch was
conducted in the Midwestern United States in 2019 [11]. These major advances in CRISPR-
Cells 2022, 11, 3471 Cas technology in maize have opened up a new era for the generation of new3 of
germplasm
12
and elite varieties.

Figure
Figure 1.1.The
Thenumber
numberof of patents
patents perper
yearyear arising
arising from from research
research on CRISPR-Cas-edited
on CRISPR-Cas-edited maize from
maize from
2015 to2021.
2015 to 2021.

3. Effective
3. Effectiveand
andPrecise
PreciseImprovement of Agronomic
Improvement TraitsTraits
of Agronomic
Since genetic variation is the basis for the improvement of crop traits, crop breeding is
Since genetic variation is the basis for the improvement of crop traits, crop breeding
aimed at developing elite varieties with high yield, good quality, and enhanced resistance
is aimed at developing
to various stresses. CRISPR-Cas elitetechnology
varieties canwith high yield,
accelerate good
the rapid quality, and
pyramiding enhanced re-
of multiple
sistance
desirable to various
traits stresses.modifying
by accurately CRISPR-Cas technology
the specific can accelerate
target genes, the the
thus forming rapid pyramiding
basis of
of
a new era in efficient and precise crop breeding [12]. The 123 patents we found deal withforming
multiple desirable traits by accurately modifying the specific target genes, thus
the basis
genetic of a new erainingrain
improvements efficient
yield,and precise
grain crop
quality, breeding
nutritional [12]. The
content, 123
plant patents we found
architecture,
flowering
deal withtime, fertility,
genetic haploid induction,
improvements andyield,
in grain environmental stress resistance
grain quality, (Figure
nutritional 2 plant
content,
and Column G of Supplementary Table S1). The total number of traits analyzed
architecture, flowering time, fertility, haploid induction, and environmental stress re- and edited
in the 123 patents was more than 123 because several patents dealt with two or more traits.
sistance (Figure 2 and Column G of Supplementary Table S1). The total number of traits
analyzed
3.1. Yield and edited in the 123 patents was more than 123 because several patents dealt
withHigh-yield
two or more traits.
maize has always been the first goal for breeders. However, the tradi-
tional breeding methods have limited effectiveness in improving maize yield, whereas the
CRISPR-Cas technique has offered unprecedented advantages in enhancing crop yield with
superior precision and speed [13]. There have been 25 patents related to the improvement
of maize yield through the CRISPR-Cas editing of different target genes, accounting for
20.33% of 123 patents. Maize yield is determined by multiple genetic factors, such as effec-
tive ear number, kernel number per ear, and 100-kernel weight, as well as by environmental
conditions [14,15]. The key genes controlling yield-related traits are, for example, ZmRLK7,
ZmEREB102, ZmCEP1, UB2, UB3, ZmCO2, and GT1, and their editing can increase maize
yield [16–18] (Supplementary Table S1). Another important way to improve maize yield
in densely planted maize is closely related to plant height and the number and angle of
leaves. For example, erect leaves are conducive to increased population density by main-
taining photosynthesis and grain filling in dense planting [13]. Thus, modifying genes that
control leaf angle, such as LG1 and ZmRAVL1, could generate new materials with compact
plant sizes and increased population yield (Supplementary Table S1) [9,19]. Therefore,
CRISPR-Cas technology provides new ways to develop high-yield germplasm resources.
CellsCells
2022,2022,
11, 11,
3471x FOR PEER REVIEW 4 of 413of 12

Figure2.2.The
Figure Thenumber
numberofofpatents
patentsrelated
related
toto the
the improvement
improvement of of maize
maize traits
traits using
using CRISPR-Cas
CRISPR-Cas tech-
technology.
nology.
3.2. Quality and Specialty Maize
3.1. Malnutrition
Yield caused by a lack of essential nutrients in cereal-based diets is a serious
threatHigh-yield
to millionsmaize
of people worldwide.
has always been the Therefore,
first goal afor
new challenge
breeders. in maize
However, thebreeding
tradi-
istional
improving themethods
breeding nutritionalhavequality
limitedof grains, including
effectiveness specific
in improving proteins,
maize yield,carbohydrates,
whereas the
CRISPR-Cas
fatty technique
acids, essential has offered
amino acids, andunprecedented
vitamins [20].advantages
In recentinyears,
enhancing crophas
progress yield
been
with in
made superior
miningprecision and speed
quality-related [13].and
genes There have been
creating new25specialty
patents related
maize to the improve-
germplasm using
ment of maize
CRISPR-Cas yield through
technology. For the CRISPR-Cas
example, we foundediting of different
25 patents target genes,
(accounting for accounting
20.33% of the
for 20.33%
total) on the of 123 patents. of
improvement Maize
maize yield is determined
quality by editing by multiple
different genetic
genes thatfactors,
encodesuch as
metabolic
effective(including
enzymes ear number, kernel numberstarch
granule-bound per ear, and 100-kernel
synthase, weight, as
starch synthase, well as
starch by envi- en-
branching
ronmental
zyme, conditions
glutamic [14,15]. The key genes
imine methyltransferase, andcontrolling yield-related
betaine aldehyde traits are, forfunctional
dehydrogenase), exam-
ple, ZmRLK7, ZmEREB102, ZmCEP1, UB2, UB3, ZmCO2, and GT1,
proteins (such as PPR protein and gliadin 20S proteasome subunit), and transcription and their editing can
factors; these patents include the creation of the new germplasm with improvedim-
increase maize yield [16–18] (Supplementary Table S1). Another important way to grain
prove maize
waxiness, yieldamino
aroma, in densely
acid planted
content,maize
folateiscontent,
closely related to plant
and starch height and and
components the num-
content
ber and angle ofTable
(Supplementary leaves. For
S1). example,
For example,erect leaves are conducive
CRISPR-Cas to increased
editing Waxy1, populationand
SHRUNKEN2,
density by maintaining photosynthesis and grain filling
ZmBADH2a/b can effectively generate waxy, sweet, and aromatic maize grain,in dense planting [13]. Thus,
respec-
modifying genes that control leaf angle, such as LG1 and ZmRAVL1,
tively [11,21,22], which may meet increasing demands for special tastes and flavors could generate new
from
materials with compact plant sizes and increased population yield (Supplementary Table
the consumer market.
S1) [9,19]. Therefore, CRISPR-Cas technology provides new ways to develop high-yield
Deficiencies in the essential vitamins and nutrients (hidden hunger) needed for op-
germplasm resources.
timal health are the main cause of many diseases (such as diabetes, cardiovascular ail-
ments, and cancer), as well as obesity. Using biotechnology in breeding provides effec-
3.2. Quality and Specialty Maize
tive strategies to alleviate hidden hunger by increasing the content of micronutrients in
crops Malnutrition causedthe
[23]. For example, by aknockout
lack of essential
of ZmGFT1 nutrients
can in cereal-basedincrease
significantly diets is athe
serious
content
threat to millions of people worldwide. Therefore, a new challenge in maize breeding is
of 5-methyltetrahydrofolate in maize, which provides a new way to alleviate folic acid
improving the nutritional quality of grains, including specific proteins, carbohydrates,
deficiency via biofortification (Supplementary Table S1). These findings and patents pro-
fatty acids, essential amino acids, and vitamins [20]. In recent years, progress has been
vide an important theoretical basis, as well as technical strategies, for the improvement of
made in mining quality-related genes and creating new specialty maize germplasm using
maize quality.
CRISPR-Cas technology. For example, we found 25 patents (accounting for 20.33% of the
Cells 2022, 11, 3471 5 of 12

3.3. Male Sterility

Heterosis is an effective way to increase crop yield, and it has been used most suc-
cessfully in breeding maize hybrids. The breeding of male-sterile lines is an important
prerequisite for heterosis utilization in crops, which can obviate the need for manual emas-
culation, reduce labor costs, and improve seed purity and yield, and thus, it has very
important breeding and commercial value [24,25] In maize production at present, most
male-sterile lines have some limitations due to susceptibility to diseases, unstable fertility,
and extremely complicated creation process. Thus, the creation and application of new
male-sterile lines is an important goal in maize production, and the identification and
functional analysis of fertility-related genes is the key prerequisite for creating new male-
sterile lines [26,27]. Recently, using CRISPR-Cas technology, a number of male-sterility
genes, encoding transcription factors, key enzymes, and other proteins, were annotated
and utilized to generate a series of new male-sterile materials in maize [25]. There are
13 patents related to the identification of fertility-related genes and the creation of new
male-sterile lines in maize using CRISPR-Cas technology (Supplementary Table S1). In
particular, based on gene editing technology, a simple method for the one-step creation of
male-sterile lines associated with the matching maintainer line (an artificially created maize
male-sterile line and an efficient transfer method) was established; this method overcomes
the bottlenecks of traditional breeding technologies and will promote the industrial applica-
tion of third-generation crossbreeding techniques [10]. Therefore, CRISPR-Cas technology
underpins new opportunities for mining male sterility genes, creating male-sterile lines,
and enhancing heterosis utilization in maize.

3.4. Stress Resistance and Herbicide Resistance

Environmental stresses such as drought, high temperature, low temperature, flooding,
pests, diseases, and soil salinity seriously affect the yield and quality of maize. Therefore, the
genetic improvement of maize yield is closely related to the enhancement of stress resistance.
Recent studies have confirmed that plant stress resistance is a quantitative trait controlled by
the interactions of multiple genes and environmental factors, and biotechnology breeding
is an inevitable choice to improve maize resistance to biotic and abiotic stresses [28].
In recent years, many key genes involved in stress responses have been identified and
used to generate new maize materials with enhanced stress tolerance with CRISPR-Cas
technology. There are seven patents related to the generation of new germplasm with
improved resistance to northern leaf blight, straw disease, rough dwarf disease, ear rot,
stem rot, and sheath blight by editing Ht1, NLB18, ZmSIZ1a, ZmSIZ1b, ZmFhb1, and
m00001d010255, respectively [29] (Supplementary Table S1). For abiotic stress, there are
nine patents related to the improvement of maize tolerance to abiotic stresses such as
drought, low temperature, and high temperature (Supplementary Table S1) by activating
the expression of ZmBG1 [30] or the knockouts of ZmEREB102, DRK, PP84, CPK2, AL14,
ZmbZIP68, ZmCIPK15, and ZmAAPa [31–33]. These studies show that the development
of new maize materials with enhanced stress resistance via CRISPR-Cas technology has
strong potential applications.
With the constantly increasing level of intensity and mechanization in maize pro-
duction, the application of herbicides is a primary and effective weed control strategy
due to their superior efficacy, reduced labor demand, relatively low cost, and increased
crop yield. In recent decades, herbicide-resistant, genetically modified crops brought a
revolution in weed management systems, with such crops becoming the most significant
transgenics [34–36]. In particular, there have been many reports of using CRISPR-Cas
technology to create desired herbicide-resistant germplasm by editing genes related to
herbicide resistance such as acetolactate synthase (ALS), acetyl-CoA carboxylase (ACCase),
and 5-enolpyruvate oxalate-3-phosphate synthase (EPSPS) in different crops, which can
facilitate the flexible use of robust, non-selective, and broad-spectrum herbicides [37,38].
In maize, two patents have reported on the new herbicide-resistant germplasm materials
created by CRISPR-Cas editing ALS1/2 and EPSPS (Supplementary Table S1), which have
Cells 2022, 11, 3471 6 of 12

good commercial prospects in maize production. Therefore, it will be of great importance

to apply this technique to identify more genes related to herbicide resistance and create
new herbicide-resistant germplasm in maize.

3.5. Plant Architecture

Maize plant architecture traits, including plant height, ear position, leaf angle, and
internode length, determine a plant’s yield by affecting the canopy structure, photosyn-
thetic efficiency, and planting density, as well as resistance to lodging and various stresses.
Thus, one of the most effective methods to increase maize yield per unit area is by breeding
elite hybrids with optimized plant architecture [39]. CRISPR-Cas technology has shown
its unique advantage in creating ideal plant-type materials in maize. There are at least
25 patents related to editing the key genes that control maize plant height and leaf angle
(Supplementary Table S1). First, plant height is closely associated with the number of
nodes and the length of each internode, and the homeostasis and signal transduction of
phytohormones such as gibberellins and brassinosteroids play important roles in the regu-
lation of plant height [40,41]. In maize, editing the key genes controlling the homeostasis
of gibberellin (ZmGA20ox3, ZmGA20ox5, ZmGA2ox3, and ZmGA3ox1), Zm-BR1, and
ZmPIF3s can quickly generate multiple dwarf plant types [42] (Supplementary Table S1).
Leaf angle is an important agronomic trait in the breeding of high-yield varieties by af-
fecting light interception, photosynthetic efficiency, and planting density [43,44]. In maize,
ZmRAVL1 regulates endogenous brassinosteroid content by activating brd1 expression,
and the knockout of ZmRAVL1 can create upright plant architecture with reduced leaf
angle and increased yield under high planting density [9] (Supplementary Table S1). These
studies indicate that CRISPR-Cas techniques can rapidly optimize plant architecture by
precisely editing the key genes, which provides an effective strategy for breeding densely
planted and high-yield varieties.

3.6. Flowering Time

Flowering is an important trait for optimum crop life cycles and yields; it marks
the transition from vegetative to reproductive growth and influences adaption to envi-
ronmental stresses. Hence, controlling appropriate the flowering time is a major goal
of breeders in developing elite varieties with better adaptations to local environmental
conditions and climatic changes [45,46]. Flowering time is a quantitative trait controlled
by multiple genes, and an increasing number of key genes from nuclear factor-Y and
the CCT transcription factor family have been identified in maize [47,48]; they are the
ideal target genes for CRISPR-Cas technology to create maize germplasm suitable for
different ecological regions. At least 17 patents (accounting for 13.82% of the total) have
reported new maize materials with improved flowering times, created by editing key
genes that control the maize flowering time, including ZmFKF1, ZmGA20ox3, ZmDTX3.1,
ZmPHYC1/2, and several CCT transcription factor genes (ZmCCT10, GRMZM2G044126,
GRMZM5G878561, GRMZM2G331652, GRMZM2G068943, and GRMZM2G172297) [49]
(Supplementary Table S1). These CRISPR-Cas manipulations provide a feasible strategy
for precisely breeding new maize varieties suitable for different environments.

3.7. Haploid Induction

Conventional breeding made slow progress in breeding maize varieties due to a long
breeding cycle, a lack of quality resources, and gene linkages/drag. Doubled haploid
technology has become an integral part of modern maize breeding due to its speed, high
efficiency, flexibility, and genetic benefits over conventional technologies, and it has huge
commercial value and major breeding prospects [50–52]. However, the genetic basis of
haploid induction remains unclear, and the selection of lines with high-frequency haploid
induction lines is difficult. Therefore, cloning new genes involved in haploid induction
is important for further elucidating its genetic mechanism and developing novel lines.
Recently, CRISPR-Cas technology was used to identify genes related to haploid induc-
Cells 2022, 11, 3471 7 of 12

tion and create high-rate haploid inducers by editing the pollen-specific phospholipase
genes [53–55]. Five patents reported that multiple haploid inducers were generated in
maize by editing ZmPLD3, ZmDMP, ZmPLA1E, ZmPLA1, and GRMZM2G471240 [53,55]
(Supplementary Table S1), suggesting that CRISPR-Cas technology will bring new oppor-
tunities and broad application prospects in the creation of maize lines with a high rate of
haploid induction.

3.8. Other Traits

Gametophytic cross-incompatibility has been used to provide reproductive isolation in
commercial maize production, and understanding its regulatory mechanism is important in
revealing the process of fertilization and improving seed production and the genetic purity
of maize products [56]. Recently, patent CN108794610B reported a new gene resource for
studying maize incompatibility by CRISPR-Cas editing ZmGa1S [57], which can be utilized
in the process of maize breeding and seed production (Supplementary Table S1).
Lodging seriously affects maize yield. Patent CN110627888B reported that the knockout of
Stiff1 by CRISPR-Cas technology can significantly increase stalk strength, thus providing new
germplasm and a method for improving maize lodging resistance [58] (Supplementary Table S1).
Moreover, two patents (CN112680472A and CN113817033A) have asserted that a knockout
of ZmELF3.1, ZmSBP20, ZmSBP25, and ZmSBP27 can create germplasm with improved
root systems and aerial root assemblages (Supplementary Table S1), providing a theoretical
basis and technical strategies for the improvement of maize root systems.
Taken together, CRISPR-Cas technology provides a powerful tool for the genetic
improvement of multiple traits in maize.

4. Ideal Target Genes Are the Key to Generating New Germplasm

To improve crop traits using gene editing technology, it is necessary to understand
the functions and molecular mechanisms of the key genes controlling specific target traits.
Selecting ideal target genes is the key to precisely creating the desired germplasm materials.
In this paper, the proteins encoded by corresponding target genes are classified according to
their function descriptions in 123 patents (Figure 3, Column F of Supplementary Table S1).
We found that 43 genes (accounting for 34.96% of the total) encode multiple members from
different transcription factor families, such as CCT, SBP, bZIP, bHLH, and TAG, whereas
20 genes (accounting for 16.26% of the total) encode various regulatory proteins, including
protein kinase, calcium-binding proteins, protein phosphatase, histone acetyltransferase,
hormone peptide, receptor protein, PcG protein, DELLA protein, and phytochrome C. It
has been reported that transcription factors and regulatory proteins participate in plant
development processes and stress responses by regulating the expression of a large num-
ber of downstream genes [59,60], making them the ideal target genes for the creation of
new germplasm through gene-editing technology. Thirty genes (accounting for 24.39% of
the total) encode enzymes such as starch synthase, galactose oxidase, dihydroflavonol-4-
reductase, lipoxygenase, gibberellin oxidase, acetyl lactate synthase, methionine synthase,
and cytochrome oxidase, which are involved in the synthesis or metabolism of specific
substances and phytohormones, cell signal transduction, redox homeostasis, grain quality,
male sterility, flowering time, plant type, and stress resistance of maize. Six genes are
involved in the post-translational modification of proteins and encoding, e.g., protein
S-acyltransferase, F-box protein, and E3 ligase, which regulate the activity, stability, and
function of specific proteins. In addition, five genes encode transporters (ATP-binding
cassette transporter, malate transporter, ABCG-type transporter, and sulfate transporter),
fourteen encode other functional proteins (guanosine diphosphate dissociation inhibitor,
membrane protein, PPR protein, gliadin, plastid ribosomal assembly factor, and glycopro-
tein), and four are associated with unknown functional proteins; these transporters and
other functional proteins play regulatory roles in multiple physiological processes and
agronomic traits. Thus, the selection of a suitable target gene is most important for the
precise creation of excellent germplasm resources using gene-editing technology.
 porter, and sulfate transporter), fourteen encode other functional proteins (guanosine di-
phosphate dissociation inhibitor, membrane protein, PPR protein, gliadin, plastid riboso-
mal assembly factor, and glycoprotein), and four are associated with unknown functional
proteins; these transporters and other functional proteins play regulatory roles in multiple
physiological processes and agronomic traits. Thus, the selection of a suitable target gene
Cells 2022, 11, 3471 is most important for the precise creation of excellent germplasm resources using gene- 8 of 12
editing technology.

Figure 3. Classification of proteins encoded by the target genes.

Figure 3. Classification of proteins encoded by the target genes.

5. Prospects of Gene-Edited Maize

5.1. Global Commercial Prospects of Gene-Edited Maize
CRISPR-Cas technology overcomes the constraints of traditional breeding methods
and provides new opportunities for ensuring world food security by precisely creating
new varieties with high yield, improved quality, and enhanced stress resistance. It has been
estimated that the market for gene editing will exceed USD 5 billion in 2025. Although gene-
edited products have been commercialized in different countries, their global marketing
also depends on the regulatory policies of each country. Without scientific consensus
and practical regulatory systems, gene-edited crops face similar situations to genetically
modified (GM) crops, and their future commercial production and public acceptance will
likely be subject to unpredictable restrictions [61]. Encouragingly, many countries, such as
the United States, Canada, Australia, Japan, Argentina, and Brazil, tend to adopt product-
oriented regulatory policies, treating gene-edited crops without foreign gene introduction
as non-genetically modified organisms (non-GMOs), generally allowing them to enter the
market without safety evaluation. Moreover, significant gene-editing policy changes have
also been enacted in Europe (https://ihsmarkit.com/research-analysis/significant-gene-
editing-policy-changes-in-europe.html, accessed on 10 May 2022). In the United Kingdom,
Parliament approves statutory instrument making gene editing trials easier to conduct
accessed on 14 March 2022. These regulatory policy changes have greatly promoted the
commercialized production of gene-edited crops, with more than 150 gene-edited crops
entering the market in the United States. Recently, CRISPR-Cas waxy corn hybrids with
agronomy superior to introgressed hybrids were generated by editing a waxy allele in
12 elite inbred maize lines [11]. Then, the USDA concluded that these waxy corns were
not to be regulated by the APHIS regulations concerning GMOs (https://www.aphis.usda.
gov/biotechnology/downloads/reg_loi/17-076-01_air_response_signed.pdf, accessed on
12 January 2018), and a limited precommercial launch was conducted in the Midwestern
United States in 2019. Subsequently, Argentina, Brazil, and Chile similarly confirmed that
CRISPR–Cas9 waxy corn is outside the scope of regulatory oversight for GMOs in these
countries [11]. In January 2022, the Guidelines for the Safety Evaluation of Gene Editing
Plants for Agricultural Use (Trial) was issued by the Ministry of Agriculture and Rural
Cells 2022, 11, 3471 9 of 12

Affairs of the People’s Republic of China, greatly simplifying the regulatory policy for
gene-edited plants; these guidelines will promote the field testing, safety evaluation, variety
certification, and commercial production of gene-edited crops in China in the future.

5.2. Opportunities and Challenges of Gene-Edited Maize in China

China has been a world leader in CRISPR-Cas-edited crops, such as rice, maize,
wheat, soybean, and others. Among the 123 global patents related to the use of CRISPR-
Cas technology in maize, there are 104 patents (accounting for 84.55%) filed from China,
documenting a large number of novel, elite germplasm materials regarding sterile lines,
haploid inducers, plant types, high yield, improved quality, and enhanced stress resistance
(Supplementary Table S1). In particular, a series of breakthroughs have been made in the
creation of third-generation male-sterile maize lines, as well as heterosis utilization and
haploid induction. Therefore, China should seize the new opportunity of gene-editing
technology to control global crop variety breeding and commercial production in the future.
Firstly, the theoretical breakthroughs and technological innovation of gene editing
in crops should be acknowledged. CRISPR-Cas technology can efficiently and precisely
create new germplasm by modifying multiple key genes that control the target traits, thus
providing new opportunities for the smart molecular breeding of crops [12]. Although
China has made fruitful achievements in the research and application of CRISPR-Cas
in crop editing, it must further develop new gene-editing technology with independent
intellectual property rights, identify important genes with breeding values, and clarify
the regulatory mechanisms governing agronomic traits. This research will provide the
theoretical basis, technical support, and key gene resources for the creation of new, elite
maize varieties.
For China, it is imperative to seize the opportunity of gene-editing technology to
promote the development and international competitiveness of the national grain industry.
Firstly, further integration of gene-editing technology with genomic selection, smart breed-
ing, and conventional breeding technologies will realize the leap from the era of molecular
breeding to the new era of intelligently designed breeding, which will accelerate the process
of breeding excellent maize germplasm.
Secondly, the application of male-sterile lines in maize breeding should be strength-
ened. The use of male-sterile lines can avoid manual or mechanical emasculation, reduce
production costs, and improve the purity and yield of seed, which has broad industrial
prospects and commercial value worldwide. With the combination of gene editing, pollen
inactivation, fluorescence seed screening, herbicide resistance screening, and conventional
breeding methods, excellent multi-control male-sterile lines will be developed in the future.
In China, 13 patents related to the creation of new male-sterile maize lines have been filed,
and it is urgent to promote their field trials and utilization in breeding.
Thirdly, gene-editing technology provides new opportunities for studying the mecha-
nism of haploid induction and the creation of haploid induction lines. Conventional maize
breeding requires continuous self-crossing for eight or more generations to produce an
inbred line, whereas haploid technology can create homozygous inbred lines over two
generations in one year, which has become one of the three core technologies of modern
maize breeding. Recently, many original studies have been conducted in China with re-
spect to mining key genes controlling haploid induction, establishing efficient systems for
haploid induction and creating haploid inducers (S1). These studies provide the theoretical
basis and technical support for promoting the transformation of maize breeding technology
in China.
It is worth noting that many reported transgenic plants have been tested for their
potential breeding value only in the laboratory or greenhouse conditions, and the lack
of field testing has resulted in the poor availability of some products [8]. In maize, the
utilization value of 1671 genes (approximately 4.4% of the genome) has been evaluated
through field tests, but only 22 genes have been proven to possess breeding values [8].
Therefore, field testing is indispensable in evaluating whether gene-edited maize can
Cells 2022, 11, 3471 10 of 12

become an important germplasm resource for breeding. Although many CRISPR-Cas-

edited maize materials have been generated, due to regulatory policies and the lack of
reliable field trials, only CRISPR-Cas-edited waxy maize hybrids have been approved for
commercial production in the United States [11]. So far, there is no report on their field
testing in China. Recently, the Guidelines for the Safety Evaluation of Gene Editing Plants
for Agricultural Use (Trial) were issued to regulate the research and field experiments
on gene-edited crops, including the regulatory framework, safety evaluations, variety
approval, and commercial production. According to these guidelines, most CRISPR-Cas-
edited maize materials that do not involve environmental safety and food security can apply
for a safety certificate in 2–3 years; then, they can be certified by the Variety Certification
Committee within 2–3 years. Thus, this system greatly accelerates the process of breeding
new varieties and their commercial production.

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/cells11213471/s1, Table S1: The patents related to the use of CRISPR-Cas in
maize.
Author Contributions: Y.W., H.Z., Q.T. and Y.K. analyzed the patent data; Y.W., H.Z., X.L. and X.W.
wrote the article. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Agricultural Science and Technology Innovation Program of
the Chinese Academy of Agricultural Sciences, the Major Projects of National Science and Technology
(2019ZX08013010-015), and the Central Public-Interest Scientific Institution Basal Research Fund
(No. 1610392022012).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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