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CRISPR-Cas9 in agriculture: Approaches, applications, future perspectives, and associated challenges

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The discovery of an adaptive immune system especially in archae and bacteria, CRISPR/Cas has revolutionized the field of agriculture and served as a potential gene editing tool, producing great excitement to the molecular scientists for the improved genetic manipulations. CRISPR/Cas9 is a RNA guided endonuclease which is popular among its predecessors ZFN and TALEN’s. The utilities of CRISPR from its predecessors is the use of short RNA fragments to locate target and breaking the double strands which avoids the need of protein engineering, thus allowing time efficiency measure for gene editing. It is a simple, flexible and highly efficient programmable DNA cleavage system that can be modified for widespread applications like knocking out the genes, controlling transcription, modifying epigenomes, controlling genome-wide screens, modifying genes for disease and stress tolerance and imaging chromosomes. However, gene cargo delivery system, off target cutting and issues on the safety of living organisms imposes major challenge to this system. Several attempts have been done to rectify these challenges; using sgRNA design software, cas9 nickases and other mutants. Thus, further addressing these challenges may open the avenue for CRISPR/cas9 for addressing the agriculture related problems.
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ISSN: 2616-1923 (Online) Malaysian Journal of Halal Research Journal (MJHR) 2020, VOLUME 3, ISSUE 1
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CRISPR-Cas9 in agriculture: Approaches, applications, future perspectives, and
associated challenges
Prabin Adhikari*, Mousami Poudel
Agriculture and Forestry University
*Corresponding Author Email: adhikariprabin8@gmail.com
Doi: 10.2478/mjhr-2020-0002
Abstract:
The discovery of an adaptive immune system especially in archae and bacteria, CRISPR/Cas has revolutionized the field of agri culture and served as a
potential gene editing tool, producing great excitement to the molecular scientists for the improved genetic manipulations. CRISPR/Cas9 is a RNA guided
endonuclease which is popular among its predecessors ZFN and TALEN’s. The utilities of CRISPR from its predecessors is the use of short RNA fragments
to locate target and breaking the double strands which avoids the need of protein engineering, thus allowing time efficiency measure for gene editing. It is
a simple, flexible and highly efficient programmable DNA cleavage system that can be modified for widespread applications like knocking out the genes,
controlling transcription, modifying epigenomes, controlling genome-wide screens, modifying genes for disease and stress tolerance and imaging
chromosomes. However, gene cargo delivery system, off target cutting and issues on the safety of living organisms imposes major challenge to this system.
Several attempts have been done to rectify these challenges; using sgRNA design software, cas9 nickases and other mutants. Thus, further addressing these
challenges may open the avenue for CRISPR/cas9 for addressing the agriculture related problems.
Keywords: nickases, endonuclease, gene editing, sgRNA binding
1.0. Introduction:
In the current scenario, world population is increasing in geometric proportion but the food materials available are increasing in arithmetic scale. By 2050,
the world population is expected to reach around ten billions; just 3 decades far away from us [1], [2]. Increasing world population along with the adverse
effect of climate change has threatened the world in terms of food security. The situation will be even exacerbated with the decrease in fertile land coupled
with reducing yields. By the recent statistics of International Rice Research Institute, it is found that with one hectare of the fertile land per 7.7 seconds is
being lost and the effect may even be highly pronounced if the accelerating rate of global temperature prevails [3]. To meet the food security in global scale,
the yield potential of the crops grown should nearly be doubled and to obtain this goal, highly adaptable crop varieties to biotic and abiotic stress should
be introduced urgently [4], [5].
The development and improvement of crop varieties has been carried out using conventional breeding techniques like hybridization and mutational
breeding which has improved crop production to some extent. Genetic manipulation technique has been used for few decades using physical, chemical and
biological mutagenesis to study the role of genes and identifying the biological mechanisms for crop improvement. Transgenic techniques have been used
further for understanding the plant biology and crop improvement. Despite using these techniques, satisfaction has never been obtained due to the presence
of some negative issues[6]. The actual yield seems to be approaching a plateau in next few years and yield stagnation has been currently reported in major
cereal crops like rice in East Asia, maize in South Europe and wheat in Northwest Europe[7], [8]. Moreover, the integration of transgene into host genome
is non-specific and a matter of public concern when it comes to the edible crop species. So, as to remove these limitations, the use of biotechnology in crop
improvement is of utmost importance [8].
In recent years, the use of sequence-specific nucleases (SSNs) has been extensively applied for precise genome editing in crop species [9]. These SSNs create
breakage of double strands in the target DNA. The DNA then gets repaired through either non homologous end joining (NHEJ) or homology-directed
recombination (HDR) pathways; the former being the more common one; resulting into insertions/deletions and substitution mutations in the target DNAs
respectively[10]. The genome editing methods offers a huge advantage in producing defined mutants unlike that of transgenic approaches with random
insertions leading to random phenotypes. Genome edited plants also carry their edited DNA for the desired traits, which offers an additional advantage[11].
These improved crops can be used in breeding programs and resulting varieties can be used directly with relatively lesser consumption issues and lesser
regulatory procedures as compared to transgenic crops/ conventional genetically modified (GM) crops[12]. This review discusses the applications of
recently advanced genome editing tool CRISPR/Cas9 in crop improvement along with the future perspective and associated challenges.
2.0. Gene editing tools: Engineered nucleases
When a non-specific nuclease domain is fused with sequence-specific DNA binding domain, then it is said to be engineered. Such fused nuclease has the
capacity to cleave the target gene and the breaks are repaired either by NHEJ or HDR, and the whole process is given the term genome editing[13]. Naturally
inspired existing technologies for genome engineering include several nucleases like Zinc finger nucleases (ZFNs), Transcription activator-like effector
nucleases (TALENs) and CRISPR/Cas9 system. ZFNs and TALENs involve tedious procedure with long turn-over time, costly, and are less reliable as
compared to the second generation CRISPR/Cas9 system[14]. More than $5000 per pair of commercial ZFNs [15]or TALENs makes it much more expensive.
Moreover, the off target activity/toxicity and base skipping activity are higher than CRISPR/cas9. Besides this, long turn-over time has made it difficult for
ZFNs and TALENs to scale up. Usually, ZFNs take several weeks to build a few pair for experts and TALENs take about a week to build a few pairs[14] which
make it more time consuming than CRISPR/Cas9[16].
Figure 1: Genome editing tools (SSNs)
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3.0. CRISPR/Cas9: An advanced genome editing tool
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a microbial adaptive immune system which was first iden tified in E.coli[17]. It
consists of two main parts: short identical repeats of 20-40 ladders and a spacer DNA which is not identical. It was known in 80’s or 90’s that the each set
of spacer DNA is unique[18], [19]. In 2000’s it was identified that the Spacer DNA matches with the viral bacteriophases[17]. At the same time the number
of genes associated with CRISPR was also identified. They are CRISPR associated or Cas genes. Cas genes make Cas proteins. Cas proteins are the helicases
that unwind DNA, and nucleases that cut the DNA. These proteins are immune system for bacteria where they could resist bacteriophases[10].
When the bacteriophase injects its DNA what normally would have happen if it does not have immune system is this DNA would hijack the cell and could
become invaded in the genome. For more importantly it would make the bunch of these bacteriophases and eventually kill the cell. Since it has a CRISPR
system what it is going to do is transcribe and translate protein. So this Cas complex and also transcribe DNA to make crRNA and it will fit right into the
protein. It is a way to fight out viral RNA that essentially breaks it apart[20]. In fact, before the infection starts, infection essentially has ended. But what
happens when CRISPR do not have spacer that matches? Well, the CRISPR-Cas system works there as well. It’s going to create a different class of protein: a
class I Cas protein, it takes the DNA in and breaks it apart, and more importantly takes that DNA and copies it into the CRISPR system[21]. CRISPR is in fact
a bunch of spacer-repeats but the spacers are essentially history of old infections that won’t be infecting again[22].
What scientists thought is if they could hijack this CRISPR system, they could perhaps use it as it is a living cell, so as to either inactivate genes or may be
even invade new genes. The research was on and CRISPR/Cas9 was developed. It is identified in labs of Jennifer Doudna and Emmanuelle Charpentier and
what she was working on was Streptococcus pyogenes and their CRISPR/Cas system. They only had one Cas protein Cas9. If we look at its major structure,
it has nucleases so as to cut the section right there. In S.pyogenes they are also creating two long strips of RNA; CRISPR RNA (crRNA) that gets fit into the
Cas and transactivating CRISPR RNA (tracrRNA) that holds the crRNA in place[19]. The whole thing forms the complex where it can break the DNA. It would
not be possible to apply if the whole system was not modified. Using one Cas9 protein but putting on the own sequence of DNA (crRNA) and connecting
these two together forms a simple system and that was what they actually did. They created a tracrRNA-crRNA chimera. They created a new type of RNA
that has a system which is really simple containing two parts; one is the Cas9 protein and the other is the chimera. In simple words, the chimera is also
called as guide RNA (gRNA)[23], [24]. CRISPR part and Cas9 part thus together forms a complex and work together. RNA that gets the information at the
target site and the protein that actually does the work of cutting.
When the viral DNA or the DNA that we want to cut enters, then it creates a guide RNA that would be complementary to the target DNA and when the DNA
starts feeding, the Double strand break (DSB) occurs (Fig2); it gets cut into two pieces, becomes inactive and fixes itself by NHEJ or HDR by the
insertion/deletions and substitution respectively. To insert the desired host DNA, host DNA is added and the DNA gets fixed[25].
Figure 1: CRISPR/Cas9 for DSBs and repair either by NHEJ or HDR
3.1. Development and modifications of CRISPR/Cas9
The most common type of cas9 protein is from the bacteria Streptococcus pyogenes (SpcAs9) which recognizes the NGC-type PAM[26]. This type of PAM
sequence is widely distributed throughout plant genomes but it actually does not cover the entire plant genome so this may increase the off target cleavage
due to complexing of gRNA with the mismatched complementary target DNA within the genome. Thus several attempts and modifications have been done
on Cas9 enzyme for achieving the target specificity and reducing the frequency of off target effects which is shown in Table 1 [8]. An increase in the
protospacer adjacent motif length is a strategy that has been used to minimize the off-target cleavage. The CRISPR/Cas system developed from the bacteria
other than S.pyogenes like Neisseria meningitidis known as Nmecas9, recognizes an 8-mer PAM sequence (5′-NNNNGATT) that can improve target specificity
and reduce potential off-target cleavage [27]while Staphylococcus aureus, Sacas9 recognizes a 6-mer PAM sequence (5′-NNGRRT; [28]. Similarly, Cas9
nuclease activity modifications have also been successful in expanding the applications of CRISPR/Cas9. First, with a modified Cas9 cleavage domain at
Cas9-D10A or Cas9-H840A and on combining with paired guide RNAs, Cas9 can cleave a targeted region on the opposite DNA strand, improving its
specificity 1001500-folds. Second, the Cas9 created by point mutation in RuvC and HNH nuclease domains called dead Cas9, or dCas9[29] or CRISPRi
become catalytically inactive and thus cannot cleave targeted regions. The co-expression of dCas9 and a specific sgRNA in the coding region of a gene can
prevent the transcription elongation process which may lead to the loss of function of incompletely translated proteins[30]. This approach can be used to
block transcription initiation by binding to the operator or the promoter of a gene, such as a transcription factor binding site or RNA polymerase binding
site. Such binding can markedly decrease gene expression. The dCas9 can be fused with different effector domains (repressor or activator) for recruiting
functional proteins to the specific genome loci, and then represses or activates the gene expression[31]. For instance, dCas9-VP64 (a transcription activator)
and dCas9-p65AD (a single copy of the p65 activation domain) can efficiently activate reporter gene expression showing that CRISPR/Cas9 can act as a
modular platform for transcription control[32]. Similarly, dCas9 can also be combined with epigenetic factors, such as histone-modifying/DNA methylation
enzymes, for the epigenetic modification of genes. Cas9 can also be fused with fluorescent protein for DNA labeling of a specific region and can be used in
chromosome imaging. Finally CRISPR/Cas9 orthologues were identified which increased the target specificity which is shown in Table 2. Distinct crRNA-
tracrRNA duplex and PAM requirements is imposed by many of the naturally occurring cas9 orthologues[30]. Hence, exploring orthologues Cas9 proteins
with similar gRNA and PAM sequences would greatly expand the possible target sequences in a given genome and thus add new Cas9 orthologues with
unique properties in CRISPR/Cas system[33]. Cas9 proteins isolated from different bacterial species had unique and expanded PAM sequences that can aid
in increasing on-target specificity. The alternative orthologous Cas9 requires different PAM sequences which increases the total number of target sites
within a plant genome. CRISPR-Cpf1 is a class II, type V endonuclease developed from Prevotella and Francisella. Cpf1 uses single guide RNA (crRNA)
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complex for the cleavage, producing cohesive ends with 4-5 nucleotides 5’ overhangs, with less or no off target events[34]. Similarly, C2C2 nuclease isolated
from Leptotrichia shahii is capable of dual nuclease activity and can target single-stranded RNA[35]. In this way, CRISPR/Cas9 can be modified using
multiple Cas9 orthologue-based platforms with different effectors like repressor, nuclease and transcription activator into the same cell, where they are
guided by specific group of guide RNAs to carry out complex gene editing [33].
Table 1: CRISPR/Cas9 Modifications and their applications
Modification
Engineering
Application
Reference
SpCas9n (Cas9n)
Substitution of aspartite to alanine (D10A) in the RuvC domain
Allows knock in via HDR
[36]
Dead cas9 (dcas9)
Cas9 nuclease inactivation and double nicking using nickase
Nicking enhances specificity
[37]
FokI Cas9 (fCas9)
Inactivated Cas9 nuclease fused with FokI nuclease
Increased on target activity
[38]
Table 2: CRISPR/Cas9 orthologues
System
Source
Protein
PAM (5′–3′)
Reference
CRISPR-cas9
Streptococcus pyogenes
Cas9
NGG
[10]
CRISPR-cpf1
Prevotella and Fracisella
Cas1, Cas2, Cas4
YTN
[34]
Ng-Ago
Natronobacterium gregoryi
Argonaute
Not required
[30]
Fig: (d)
Fig: (b)
Fig: (e)
Fig: (a)
Applications
of
CRISPR/Cas9
Fig: (c)
Fig: (f)
Figure 3: Applications of CRISPR/Cas9 [Fig(a):Genome editing using CRISPR/Cas9 through common pathway of DSB repair; Fig(b):Multiplexing using
CRISPR/Cas9; Fig (c): Gene knock-in/replacement; Fig (d): Gene knockout using CRISPR/Cas9; Fig (e): Base editing; Fig (f) :High throughput mutant library]
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4.0. Applications of CRISPR/Cas9 in crop improvement
The simple, efficient and highly specific CRISPR/Cas9 system is a promising tool for genome editing and is expected to have larger impacts on plant biology
and also on crop breeding. The elite cultivars can be precisely modified using genome editing technique, saving the time required compared to backcrossing
in conventional breeding programs[39]. With the multiple traits being modified at a time, CRISPR/Cas9 system provides an efficient approach to pyramid
breeding[40]. Gene editing by CRISPR/Cas9 has been adopted in nearly 20 crop species so far[41] for various traits including yield improvement, and biotic
and abiotic stress management. NHEJ mediated gene editing is the most direct application of CRISPR/Cas9 gene editing. Biotic stress imposed by pathogenic
micro-organisms account for more than 42% of potential yield loss and contributes to 1 5% of global declines in food production[42]. The negative
regulators of disease resistance and grain development can be knocked out to obtain greater yield, host resistance against targeted pathogens and abiotic
stresses like drought and salinity. In addition, CRISPR/Cas9 provides alternative approaches for delivering target genes into crops without any transgenic
footprint, such as by viral infection, agro-infiltration, or preassembled Cas9 protein-sgRNA ribonucleoproteins transformation so as to bypass the
traditional regulations on genetically modified organisms[43].
Table 3: Applications of CRISPR/Cas9 in agricultural crops
Crop
Target gene
Target trait
References
Rice
LAZY1
Tiller-spreading
[43][50]
[51]
[52]
[45]
[53]
[54]
[49]
[55]
[56]
[57]
Gn1a, GS3, DEP1
Enhanced grain number, larger
grain size and dense erect
panicles
SBEIIb
High amylose content
OsERF922
Enhanced rice blast resistance
OsSEC3A
Resistance to rice blast causing
pathogen Magnaporthe oryzae
OsSWEET13
Bacterial blight resistance
OsMATL
Induction of haploid plants
ALS
Herbicide resistance
EPSPS
Herbicide resistance
25604 gRNA for 12802 genes
Creating genome wide mutant
library
OsPDS, OsMPK2, OsBADH2
Involved in various abiotic
stress tolerance
OsMPK2, OsDEP1
Yield under stress
OsDERF1, OsPMS3, OsEPSPS,
OsMSH1, OsMYB5
Drought tolerance
OsMPK5
Various abiotic stress tolerance
and disease resistance
OsAOX1a, OsAOX1b,OsAOX1c,
OsBEL
Various abiotic stress tolerance
OsHAK-1
Low cesium accumulation
OsPRX2
Potassium deficiency tolerance
Wheat
TaMLOA1, TaMLOB1 and
TaMLOD1
Resistance to powdery mildew
[58]
[45], [59], [60]
[61]
GW2
Increased grain weight and
protein content
EDR1
Powdery mildew resistance
TaGW2
Incresase seed size
Maize
Wx1
High amylopectin content
[62], [63]
TMSS
Thermosensitive male sterile
ALS
Herbicide resistance
ARGOS8
Drought stress tolerance
Tomato
SIMLO1
Powdery mildew resistance
[8], [46]
SIJAZ2
Bacterial speck resistance
SP5G
Earlier harvest time
SIAGL6
Parthenocarpy
SP, SP5G, CLV3, WUS, GGP1
Tomato domestication
Potato
ALS
Herbicide resistance
[29]
Wx1
High amylopectin content
[64]
Mushroom
PPO
Anti-browning phenotype
[65]
Grapefruit
CsLOB1
Citrus canker resistance
[66]
CsLOB1 promoter
Alleviated citrus canker
[66]
Orange
CsLOB1 promoter
Citrus canker resistance
[67]
Cucumber
eIF4E
Virus resistance
[68]
Camelina sativa
FAD2
Decreased polyunsaturated
fatty acids
[69]
Soybean
ALS
Herbicide resistance
[70]
Flax
EPSPS
Herbicide resistance
[71]
Cassava
EPSPS
Herbicide resistance
[72]
4.1. Knockout mediated crop trait improvement
Eliminating the negative traits that confer undesirable effects to the crop needs to be knocked out so as to obtain greater yield and biotic and abiotic stress
tolerant crop variety. Hybrid breeding techniques and other important aspects of the crop breeding has been improved by knockout mechanism of gene
editing[73].
4.1.1. Increasing yield
To cope up with the widespread food insecurity, the major sector to be think upon is crop yield. Yield is a complex trait governed by many factors. Knocking
out negative regulators known to affect yield-determining factors such as grain number (OsGn1a), grain size (OsGS3), grain weight (TaGW2, OsGW5, OsGLW2,
or TaGASR7), panicle size (OsDEP1, TaDEP1), tiller-spreading (LAZY1)and tiller number (OsAAP3) created the expected phenotypes in plants with loss-of-
function mutations in these genes[44], [45], [74]. Simultaneous knockout of three grain weightrelated genes (GW2, GW5, and TGW6) in rice led to trait
pyramiding, which greatly increased grain weight[75]. M. Li et al., (2016) mutated the Gn1a, DEP1, and GS3 genes of the rice cultivar Zhounghua11 using
CRISPR/Cas9, producing mutants with larger grain size, dense erect panicles and enhanced grain number. When Grain Weight 2 (GW2) gene in wheat is
disrupted, it resulted in increased grain and protein content in wheat[77, p. 2]. However, because most yield-related traits are quantitative and controlled
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by quantitative trait loci, simply knocking out individual factors may not be sufficient to increase yield in the field. Huang et al., (2018) recently discovered
a technique for large scale identification of genes that contributes to complex quantitative traits by combining pedigree analysis, whole-genome sequencing,
and CRISPR/Cas9 technology[78]. The author sequenced 30 cultivars of the rice variety IR8 and selected 57 genes from high-yielding lines for gene editing
via knockout or knockdown using Cas9 or dCas9. This provided insight in crop yield development and may facilitate molecular breeding in rice[79].
4.1.2. Increasing quality
Crop quality can be improved by genome editing techniques. Till date, quality improvements by genome editing have been done for traits such as fragrance,
longer keeping quality, improving oleic acid content, starch content etc. Fragrance is an important quality trait in rice, and fragrant rice varieties when
cooked have increased commercial value. A defect in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the biosynthesis of 2-acetyl-1-
pyrroline, the major fragrance compound in fragrant rice. With the advent of convenient CRISPR/Cas9 techniques, scientists have recently added the
fragrance trait to more than 30 elite rice cultivars in major planting areas of China [73]. CRISPR/Cas9 technology has been used to target the FAD2 gene in
Camelina sativa, the emerging oil seed plant, to improve oleic acid content while decreasing polyunsaturated fatty acids[69]. Gluten proteins from cereal
crops which trigger celiac disease are formed by α-gliadin gene family. The CRISPR/Cas9 genome editing tool offers a new way to alter traits controlled by
large gene families with redundant functions. Indeed, by knocking out the most conserved domains of α-gliadin family members, researchers have created
low-gluten wheat[80]. CRISPR/Cas9 has been used in rice to generate the targeted mutations in SBEIIb, leading to higher proportion amylose, which
improved the nutritional content and fine structure of starch [47]. Waxy maize with improved digestibility and higher bio-industrial application is prepared
by DuPont Pioneer by knocking out the maize waxy gene (Wx1), which encodes Granule-bound starch synthase (GBSS) gene responsible for making
amylose[81]. Development of parthenocarpic tomato fruits with huge market potential in processing industry was achieved simultaneously by two different
groups. Klap et al., (2017) carried out knockout of Slagamous-like 6(SlAGL6) gene that otherwise severely hamper fertilization-dependent fruit set; made
mutant plants capable of producing parthenocarpic fruits under heat stress conditions[82]. Alternatively, the other group has obtained parthenocarpic
tomato fruits by mutating SlIAA9 gene involved in auxin signaling pathway that suppress the parthenocarpy[83]. By knocking out the gene responsible for
polyphenol oxidase enzyme synthesis using CRISPR/Cas9 [65] developed a non-browning mushroom which triggers the post harvest quality and fetch
higher prices.
4.1.3. Increasing biotic and abiotic stress tolerance
The yield loss caused by the disease causing pathogen and other abiotic stress is significantly higher. According to Ficke et al., (2017), the estimated yield
loss due to plant pathogens is up to 16%[84]. So many attempts have been made in molecular biology to make disease resistant varieties. Y. Wang et al.
(2014) has simultaneously edited three homoeoalleles TaMLOA1, TaMLOB1 and TaMLOD1 that confer heritable resistance to powdery mildew[85]. The
CRISPR/Cas9 technology is used to generate Taedr1 wheat plants by simultaneous modification of the three homeologs of EDR1 which resulted in plants
resistant to powdery mildew[86]. In rice, resistant varieties against blast disease and bacterial blight were obtained separately by mutagenesis of OsERF922,
OsSEC3A and OsSWEET13 genes[56], [87], [88]. Further, powdery mildew and bacterial speck resistant tomato varieties were obtained by editing SlMLO1
and SlJAZ2 respectively[89], [90]. By the modification of CsLOB1 promoter, canker symptoms were alleviated in Duncan grapefruits[66]. The technology
was further used to disrupt the coding region of CsLOB1 resulting in no canker symptoms in Duncan grapefruit[67]. Chandrasekaran et al. (2016) conducted
a research in cucumber by disrupting eIF4E (Eukaryotic translation initiation factor 4E), broad virus resistance was developed[68]. The plants were seen
immune to cucumber Vein Yellowing virus (Ipomovirus) and were also resistant against potyviruses, Zucchini yellow mosaic virus and Papaya ring spot
mosaic virus-W. CRISPR/Cas9 has also produced tungro disease resistant eif4g rice[91] and cotton leaf curl disease-resistant clcud cotton[92]. The
destructive insect pests of rice i.e. plant hoppers and stem borers are the major yield reducing factors in rice. It was found that disrupting the OsCYP71A1
blocked serotonin biosynthesis and greatly increased salicylic acid levels, thereby confer resistance against these pests[93].
By developing slnpr1 mutant from isolated SlNPR1 gene of tomato ‘Ailsa Craig’ using CRISPR/Cas9 resulted into drought tolerance in tomato[94].
Modification of OsPDS, OsMPK2, OsBADH2 genes in rice had led to increased tolerance to abiotic stresses[52]. Similarly Zhang et al. (2014) identified that
modifications on OsDERF1, OsPMS3, OsEPSPS, OsMSH1, OsMYB5 genes lead to the drought tolerant varieties in rice[53]. Similarly, OsHAK-1 and OsPRX2 gene
were edited in rice for obtaining low cesium accumulation and potassium deficiency tolerance respectively[55], [57]. CRISPR/Cas9 has been used to study
the role of genes. Plant annexins which play a significant role in stress tolerance and plant development were tested using CRISPR/Cas9. Rice annexin gene
(OsAnn3) was tested for its role in cold stress by OsAnn3 CRISPR knockouts [95].
4.1.4. CRISPR/Cas9 for hybrid breeding
An essential requirement for a high-quality hybrid variety is a male-sterile maternal parent. Using CRISPR/Cas-mediated gene knockout, tremendous
progress has been made to produce male sterile lines. The thermosensitive genic male sterile 5 gene (TMS5) was knocked out in maize to generate male
sterile line[96]. In similar ways thermosensitive male-sterile tms5 lines were developed in rice[97, p. 5], photosensitive genic male-sterile csa rice[98] and
ms45 wheat were developed[99]. Recently, haploid rice was obtained by knockout of OsMATL by CRISPR/Cas9[100]. The selfish-gene suicide mechanism
in rice caused by the toxic ORF2 gene was knocked out improving the fertility of japonica-indica hybrids[101].
Similarly, CRISPR/Cas9 can be used by the breeders for domestication of wild varieties. Wild tomato accessions were introduced by targeting the coding
sequences, cis-regulatory regions, and upstream open reading frames of genes associated with tomato morphology, flower and fruit production and
ascorbic acid synthesis[102].
4.2. Gene insertion and replacement
CRISPR/Cas9 mediated gene insertion and replacement has mainly been used for developing herbicide resistant crop varieties. Substitution of key amino
acids in the conserved domains of the endogenous acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) can confer
resistance to sulphonylurea-based herbicides[73]. Herbicide resistant rice variety was developed by utilizing several methods like disrupting DNA ligase 4
that is implicated by NHEJ repair[103], NHEJ mediated intron targeting[104], using two single-guide RNAs (sgRNAs) targeting the repair template [48] and
the use of chimeric single-guide RNAs (cgRNAs) carrying both repair template sequences and target site[105]. Butler et al. (2016) produced herbicide-
resistant potatoes using geminivirus replicons that increase the copy number of CRISPR/Cas9 and a repair template[64]. Similarly, herbicide-resistant flax
has been generated using a combination of single-stranded oligonucleotides and CRISPR/Cas9[71]. Recently, glyphosate tolerance variety of cassava was
generated by a promoter swap and dual amino-acid substitutions achieved at the EPSPS locus[72]. In maize, grain yield under drought stress condition was
increased by editing ARGOS8 gene[62]. When the GOS2 promoter was inserted into the 5’- untranslated region of the native ARGOS8 gene, or when it
replaced the ARGOS8 promoter, increased ARGOS8 transcripts were detected that resulted in increased drought tolerance.
5.0. Novel breakthroughs
5.1. Application in Base editing
Many agronomically important traits are conferred by single-nucleotide polymorphism either in coding or non-coding sequences. This makes base editing
quite useful in plant breeding and crop improvement. Genome wide-association studies have shown that single-base changes are responsible for the
variations in elite traits in crops[106]. Hence, techniques for precise point mutations in crops are needed urgently. CRISPR/Cas9 mediated genome editing
tool can be efficiently used for accurately converting one DNA base into another, without the use of a DNA repair template[107]. Base editing techniques
requires the use of Cas9 nickase (ncas9) or dead cas9 (dcas9) fused to an enzyme with base conversion ablity. For example, cytidine deaminases convert
cytosine to uracil which is treated as thymine in DNA repair or replication process, creating a C-G to T-A substitution[107], [108]. Likewise, adenine
deaminases convert adenine to inosine, which is treated as guanine by polymerase, creating A-T to G-C substitutions[109]. Adeninne-deaminase-mediated
base editing (ABE) is more complicated that Cytidine-deaminase-mediated base editing as no known naturally occurring cytidine deaminases catalyze
adenine deamination in DNA, rather than RNA, which was later made more efficient by the team of Gaudelli using several rounds of directed evolution and
protein engineering[109].
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One of the applications of base editing is to confer herbicide resistance. Sulfonylurea- or imidazolinone-resistant rice[46], Arabidopsis[110], and
watermelon[111] have been generated by targeting ALS with a plant cytidine base editor. CBE can also be used to produce nonsense mutations that disrupts
the gene of interest and knockout their functions.CBE is highly specific than conventional SSN-mediated knockout, causing few if any indels[108]. Haloxyfop-
R-methyl resistant rice has been created by targeting acetyl-coenzyme-A carboxylase ACCase gene with a plant adenine base editor [112]. Using ABE,
fluorescence-tracking A to G base editor has been generated in rice[113]. Likewise, multiplex base editing was carried out using adenine base editor in
rice[114]. In this way, base editing tool has given genome editing a new dimension, broadening its applications with nucleotide specific modifications at
specific genomes.
5.2. Transgene-free genome editing
Conventional genome editing involves the delivery and integration of DNA cassettes encoding editing components into host genomes. The DNA cassettes
then get degenerated but the fragments get incorporated which may produce undesirable effects[115]. The introduction of foreign DNA raises the
regulatory issues regarding GM organisms. Side by side prolong expression of genome editing tools leads to the more off target activity due to the abundant
nucleases in these organisms. This nature of conventional genome editing requires the introduction of DNA free genome editing tool which reduces the off
target activity as well as escape the regulatory concerns from the national and international agencies[108]. DNA free genome editing was first conducted
in Arabidopsis, tobacco, lettuce, and rice by transfection of CRISPR/Cas9 ribonucleoproteins (RNPs) into the plant protoplast[43]. Unfortunately, efficient
and regenerable protoplasts system are not available for most of the agricultural crops which led to the particle bombardment mediated DNA free genome
editing methods. CRISPR/Cas9 RNA and CRISPR/Cas9 RNPs have been delivered to wheat embryo by particle bombardment methods for obtaining genome
editing[116], [117]. CRISPR/Cas9 RNPs has been used in maize not only to knockout mutants, but also to obtain targeted knock in mutants with the help of
single-stranded DNA oligonucleotides[63].
5.3. Multiplex Genome Editing using CRISPR
The simultaneous targeting of several genes using a single molecular construct is multiplexing; which is the major advantage of CRISPR/Cas9 over its
predecessors. Multiplexing has been reported in several crops by the assembly of multiple gRNAs under the control of a U3 or U6 promoter into a single
construct[74],[118]. More recent gene editing methods involve exploitation of self cleavage capacity of RNA molecules containing tRNA sequences. The
alternating sgRNA and tRNA sequences under the control of single U3 or U6 promoter permits reduction in size of the construct and thus limits the risk of
silencing due to direct repetition of promoter sequences. The Polycistronic tRNA-gRNA (PTG) system uses such strategy to generate hereditable mutation
in TaLpx-1 and TaMLO genes in hexaploid wheat[61]. The editing efficiency of PTG system was validated in wheat which makes it an effective tool for rapid
trait pyramiding. Alternative to PTG systems, MGEs has been reported in rice, where crRNA transcription was obtained from introns inserted into Cpf1 and
Cas9 sequences[119].
5.4. High-throughput plant mutant libraries
Whole genome scale library is an important tool for functional genomics. CRISPR/cas9 generated knockout mutant libraries has been constructed, where
they targeted nearly 13,000 genes that are highly expressed in rice shoot base tissue and obtained more than 14,000 independent T0 lines[120]. Similarly,
34,234 genes in rice were targeted by another group and generated more than 90,000 transgenic plants[121]. Later on, immunity-associated leucine-rich
repeat genes of subfamily XII, comprising 54 members in tomato were produced[122].
6.0. Challenges regarding CRISPR/Cas9 tool
Although genomes of many crop plants have been sequenced, the function of the vast majority of genes remains unknown. In othe r words, we have not
reached the level of complete understanding of the functions of the major genes in agricultural crops. However, recent genome sequencing technology and
genome-wide association studies (GWAS) allow us to predict the function of many genes, which confer resistance to biotic and abiotic stress, increase yield,
quality etc. in cultivated crops. The field test after gene editing is necessary for correct evaluation of agronomic fitness. Most of the genes may have dual
role in plant and may affect the physiology of plant along with altering the life cycle of pathogen, for example tri ple knockouts of wheat TaMLO were not
only resistant to powdery mildew but also showed leaf chlorosis[85].
6.1. Low efficiency of HR
Genome editing in the type-2 mode presents a considerable technical hurdle for regulators and ironically will probably be the most useful for plant breeding
because it enables editing of existing alleles to redefine their function or new ones to be ‘knocked-in’. NHEJ is the dominant DSBs repair mechanisms in
many plant cell types and results in rapid DSBs repair or imprecise genome alteration. The delivery of repair templates is required in large amount by HDR
to outcompete NHEJ which has always been a challenge. The delivery of DNA repair template has been improved using DNA replicons (deconstructed
geminiviruses) but it has not been a perfect solution[31]. In that case an HR repair system, like Genome Repair Oligonucleotide technology, HDR boosters
and NHEJ inhibitors are required[123], [124].
6.2. Off-target effects
When the CRISPR/Cas9 complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target
DSB and cause non-specific genetic modifications[36]. High frequency of off-target activity (≥50%) of RGEN (RNA-guided endonuclease)-
induced mutations at sites other than the intended on-target site is one major concern[125].
The most common type of cas9 protein is from the bacteria Streptococcus pyogenes (SpcAs9) which recognizes the NGC-type PAM[26]. This type of PAM
sequence is widely distributed throughout plant genomes but it actually does not cover the entire plant genome so this may increase the off target cleavage
due to complexing of gRNA with the mismatched complementary target DNA within the genome. Moreover, the CRISPR/Cas9 system accepts at least three
mismatches in the 20 bp DNA target sequence which increases the off target activity.
6.3. Delivery of CRISPR/Cas9
The greatest challenge to the implementation of CRISPR/Cas9 in agriculture is effective delivery of CRISPR/Cas9 machinery to right plant cells and
subsequent regeneration or expression of viable plants. Traditional plant transformation systems including tissue culture and gene transfer remain
preferred method for delivery but these are time-consuming, labour-intensive and produce random somatic mutations which renders inefficient[31].
Moreover, many crops are recalcitrant to the regeneration through tissue culture. This creates an urgent need of novel delivery systems to achieve high
efficiency genome editing in plants which may include use of regeneration boosters to enable tissue culture in recalcitrant species or even direct delivery
to plant apical meristems or pollen grains to obtain edited plants without tissue culture[126].
Among all the challenges, public acceptance gets the greatest priority especially in case of agricultural commodities. As the foreign DNA may impose some
side effects, modified crops have been regulated by the regulating authorities hindering the growth of CRISPR system.
7.0. Future prospects
Although much progress has been made in CRISPR/Cas9-based genome editing technology in the last few years, there are still some challenges left out: off-
target effects, side effects on nearby genes, mechanisms underlying the different effects of different sgRNAs on mutation efficiency, methods for efficient
delivery in polyploid plants and regulatory concerns. Despite these challenges, the CRISPR based system will surely revolutionize and overcome most of
the challenges. With the tremendous enthusiasm of the research community, genome editing technologies as represented by the CRISPR/Cas9 system will
improve rapidly. This simple, affordable, and elegant genetic scalpel is expected to be widely applied to enhance the crop performance in the near future[32].
The CRISPR/Cas system has a great potential for improving the plant designs and exploit the potential of plant synthetic biology by inserting artificial DNA
sequences, including promoters, genes, transcriptional regulatory elements and genome assemblies into the plant genomes. Nitrogen is a critical limiting
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factor for crop growth and development. CRISPR/Cas system could be used to transfer the genetic elements of the Nod factor signaling pathway from the
legumes to cereals allowing the cereal crops to fix atmospheric nitrogen, which reduces our dependence on inorganic fertilizers.
Several attempts have been made for improving the environmental and disease tolerance of already domesticated crops. In stead, plant species that are
already well adapted to different environments could be domesticated with high value traits. For example, the start-up Arvegenix is using the CRISPR/Cas
tool to improve oil and meal quality in winter annual plant field pennycress. The goal is to make pennycress a cover crop that can be planted between two
main growing seasons and a product similar to canola for oil and feed industry[31]. Next example of domestication by genome editing is tomato. A wild
relative of tomato called ground cherry was edited and produced plants with higher yield and bigger fruit[127]. In the future, agro-biodiversity can be
achieved and many problems associated with sustainable agriculture can be solved with the domestication of new crops with increased tolerance to a range
of challenging environments, including deserts, maritime regions, low-nutrient soil, and cold climates.
Effective delivery of CRISPR/Cas9 machinery has always posed a major challenge to the CRISPR/Cas9 system. Current delivery systems are limited to
specific plant species, genotypes and tissues. Thus, improved agrobacterium-mediated transformation may expand the range of delivery system[128].
Beside this, almost all the present delivery systems use the laborious and time consuming tissue culture techniques. This req uires the further innovation
in achieving genotype-independent, tissue culture-free delivery via the plant germline or meristematic cells[129]. For example, use of pollen mediated
transfer will overcome the limitations of species specificity and regeneration using pollination or artificial hybridization. Novel delivery systems using the
nanotechnology like carbon nanotube mediated delivery also have great potential in expanding the applications of CRISPR/Cas9 as they may cause little
cellular damage, have low toxicity and yield higher transformation efficiencies[130].
Off target activity of CRISPR/Cas9 is one of limitations hindering its applications. Thus several attempts and modifications have been done on Cas9 enzyme
for achieving the target specificity and reduce the frequency of off target effects. Modification of cas9 by the cas9 nuclease inactivation and double nicking
using nickase enhances the specificity[37]. Likewise, inactivated Cas9 nuclease fused with FokI nuclease also increases on target activity[38]. An increase
in the protospacer adjacent motif length is a strategy that has been used to minimize the off-target cleavage. Improving the specificity of Cas9-linked base
editors by extending sgRNA guide sequences, linking APOBEC1 with Cas9-HF1, and delivering base editors via RNPs could increase the applications of
CRISPR/Cas9[131], [132].
Gene regulation, protein domain swaps and even new gene functions can be achieved in different scales with SDN-2 or SDN-3. Although, low frequency of
HDR presents a considerable technical hurdle, increased efficiency can be obtained by the use of DNA replicons (deconstructed geminiviruses), but there
is no clear indication of forthcoming breakthrough. Furthermore, Agrobacterium mediated delivery that uses type IV secretion system to deliver virulence
effector proteins to plant cells and VirD2 protein covalently linked to the single-stranded T-DNA allows T-DNA transfer through protein transfer
apparatus[133]; this mechanism could co-deliver CRISPR DNA or RNP with donor templates that stimulate HDR mediated genome editing. In mammalian
cells simultaneous knockout of PolQ and genes essential for classical NHEJ (Ligase4, Ku70, Ku80) resulted in increased efficiency of HDR-mediated gene
editing[134]. Same type of NHEJ inhibitors and HDR boosters can be used in plants too.
Target specific modifications using DNA free genome editing method could be effective so as to obtain non transgenic crops with transgenic method. The
so obtained product could pass the regulatory concerns and gain public trust which increases the application of CRISPR in agr iculture. Till now, what has
been achieved with CRISPR technologies is just the tip of the iceberg. A sustainable future for agriculture can now be imagined using this new powerful
genome editing tool. With that comes a responsibility to continue to resolve both the scientific and public concerns regarding its usage for a sustainable
future[31].
8.0. Conclusion and Recommendations
Increase in world population from 7.3 billion to 9.7 billion by 2050 accompanied with increasing climate change has exacerbated the problem of food
shortage. At the same moment, CRISPR/Cas9 has come at the right time when the conventional agriculture is striving to meet the growing food demands.
This potential genome editing tool has provided scientists the ability to precisely and quickly insert the desired traits than other conventional breeding
techniques. Application of genome editing tools in crop improvement to increase the yield, quality, biotic and abiotic stress tolerance and other traits will
be a prominent area of work in the future. In the last 5 years, it is being applied vigorously in crops for functional genomics studies and combating biotic
and abiotic stresses as well as to improve other important agronomic traits. Though several modifications to this technology have led to increase on-target
efficiency, most work carried is preliminary and needs further improvement. Nevertheless, CRISPR/Cas9 based genome editing will gain popularity with
time and be an essential technique to obtain ‘suitably edited’ plants that will help achieve the zero hunger goal i.e. second SDGs and maintain feed to the
growing human population. Thus, advance molecular tool like CRISPR should be used in massive scale so as to make the world combat the global food
challenges. Further researches should be done to overcome the challenges associated.
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... In orphan crops cassava and flax herbicide resistance has been introduced by targeting a gene EPSPS (Sauer et al., 2016;Hummel et al., 2018); whereas ALS was targeted in soybean (Cai et al., 2015). Similarly, many traits have been introduced or improved by targeting various genes in some economically important crops plants such as maize, tomato, potato, grapes, orange, cucumber, tea, etc. (Adhikari and Poudel, 2020;Bhatta and Malla, 2020). ...
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Abstract Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.
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Crop improvement by inbreeding often results in fitness penalties and loss of genetic diversity. We introduced desirable traits into four stress-tolerant wild-tomato accessions by using multiplex CRISPR–Cas9 editing of coding sequences, cis-regulatory regions or upstream open reading frames of genes associated with morphology, flower and fruit production, and ascorbic acid synthesis. Cas9-free progeny of edited plants had domesticated phenotypes yet retained parental disease resistance and salt tolerance.
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Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits. Thus, translating knowledge from model crops enables rapid creation of targeted allelic diversity and novel breeding germplasm in distantly related orphan crops.
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Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade‐off is a major challenge for obtaining broad‐spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co‐receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by Pto DC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co‐receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C‐terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to Pto DC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in SljazΔjas plants. Our results solve the defense trade‐off in a crop, by spatially uncoupling the SA‐JA hormonal antagonism at the stomata, entry gates of specific microbes such as Pto DC3000. Moreover, our results also constitute a novel CRISPR/Cas‐based strategy for crop protection that could be readily implemented in the field. This article is protected by copyright. All rights reserved.
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The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (e.g., Nmcas9, Sacas9, and Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.
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Intraspecific haploid induction in maize (Zea mays) is triggered by a native frameshift mutation in MATRILINEAL (MATL), which encodes a pollen-specific phospholipase. To develop a haploid inducer in rice (Oryza sativa), we generated an allelic series in the putative ZmMATL orthologue, OsMATL, and found that knockout mutations led to a reduced seed set and a 2–6% haploid induction rate. This demonstrates MATL functional conservation and represents a major advance for rice breeding. A MATL gene mutation was found to induce haploids in maize. Now, knocking out the MATL orthologue in rice results in haploid induction at a rate of 2–6%, suggesting the functional conservation of MATL, and represents an advance for rice breeding.
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