*6.3. Crop Yield and Quality Improvements via CRISPR*/*Cas9*

Two other important agricultural traits are crop yield and quality that need to be improved through the CRISPR/Cas9 system to ensure food security worldwide (Table 5). Crop yield is a complex, multi-genic, and quantitative trait that is influenced by several features. The CRISPR/Cas9 technology has demonstrated its worth for quick yield improvement in crops.

In many studies, the CRISPR/Cas9 technique has been used to knockout the genes that negatively regulate yield related-traits including tiller number (*OsAAP3*), panicle size (*OsDEP1*, *TaDEP1*), grain weight (*TaGW2*, *TaGASR7*), grain size (*OsGS3, OsGRF4*), and grain number (*OsGn1a*). The results demonstrated that CRISPR/Cas9 is an efficient technology for improving crop yield [236–240]. A multiplexing GE strategy has been employed to mutate three genes simultaneously including *GS3*, *GW2*, and *GW5*, and *TGW6* which headed towards trait pyramiding and enhancing grain size and weight in rice [241]. Similarly, Li and coworkers applied the CRISPR/Cas9 system to knockout three yield-related genes, *Hd2*, *Hd 4* and *Hd5*, which resulted in early heading in rice [242]. It was reported that the *OsSWEET11* gene has a crucial role in grain filling and sucrose transportation. So, the CRISPR/Cas9 system was applied to disrupt the *OsSWEET11* gene, which led to decreased sucrose concentration and reduced grain weight. This study suggested that the overexpression of this gene may be beneficial for maximizing rice yield [243]. In wheat, CRISPR/Cas9-mediated GE knockout of the *GASR7* gene increased kernel weight [148]. Recently, a new approach has been established for gene identification on a large-scale that assists in examining the complex quantitative traits, including yield, by integrating the CRISPR/Cas9 tool, whole genome sequencing, and pedigree analysis. In a study, 30 varieties of "Green Revolution miracle rice" were subjected to genome sequencing and 57 genes controlling yield-related traits were screened. Knockout mutants of those 57 genes were created using the CRISPR/Cas9 technique. Phenotyping indicated that several genes are crucial for regulating yield-related traits in rice [244].

There are also many applications of CRISPR/Cas9 technology for quality improvement in crops such as storage quality, nutritional value, fragrance, and starch contents. For example, the cooking and eating quality of rice has been improved by mutating the *Waxy* gene using CRISPR/Cas9 [245]. The nutritional value of rice has also been improved by knocking out the *SBEIIb* gene which resulted in more amylose synthesis [246]. Similarly, the starch synthase gene *GBSS* was mutated via CRISPR/Cas9 in potato. The mutated lines showed decrease levels of amylose and enhanced the concentration of the amylose/amylopectin ratio [152]. In 2018, Sanchez and colleagues, carried out CRISPR/Cas9-mediated GE of the gluten-encoding gene family α*–gliadin* to produce low-gluten wheat [247]. To improve oil composition of soybean, the CRISPR/Cpf1 system was employed to disrupt the *FAD2-1B* and *FAD2-1A.* The results revealed high-yielding soybean plants with improved levels of oleic acid [163]. Moreover, it was reported that the zein protein has been reduced by 12.5% in kernels by disrupting the *PPR* and *RPL* genes in maize. The mutated plants indicate increased production levels of healthy tryptophan and lysine in maize [182]. Sorghum nourishment quality has been improved by targeting *k1C* genes which were responsible for poor digestibility and hindered production of important amino acids [248]. Recently, some other studies for quality improvement have been carried out via the CRISPR/Cas9 system, such as *Brassica napus* with high oleic acid concentration [249], long shelf life of tomatoes [250], and increased lycopene levels in tomato [251].

In summary, the above described studies reveal that CRISPR/Cas9-mediated modern breeding techniques can be utilized to attain valuable mutations for improving crop yield and quality.


**Table 5.** Summary of CRISPR/Cas9 applications in major crops for yield and quality improvement.

PRUNING 5G (*SP5G*), Growth-regulating factor 4-like (*GRF4*), Glutathione synthetase (GS3), Protein STRICTOSIDINE SYNTHASE-LIKE 10 (*TGW6*), Cytokinin dehydrogenase 2-like (*Gn1a*), Keratin-associated protein 5-5 (*DEP1*), Ubiquitin-protein ligase (*GW2*), GA-induced protein (*GASR7*), Senescence-inducible chloroplast stay-green protein 1 (*SGR1*), Lycopene epsilon-cyclase (*LCY-E*), Glycoside hydrolase family 13 protein (*SBEIIb*), Granule-bound starch synthase (*GBSS*), 2-oxoglutarate-dependent dioxygenase 2 (*GAD2*), Pentatricopeptide repeat (*PPR*), Ribosomal protein lateral (*RPL*).

### **7. Regulatory A**ff**airs of Genome-Edited Crops**

Since the development of the first genetically modified organism (GMO) in 1995, firm rules and sanctions have been imposed to regulate GM crops worldwide. In most European countries, GM crops are still banned for commercial production and release of GM crops in the field and consumer market is prohibited. Similarly, GE non-transgenic crops may also be banned if regulatory bodies consider them as GMOs. GM crops continue to provoke extensive public misunderstanding and mistrust despite 22 years of commercialization and cultivation on 189.8 million hectares in 2017 with approximately US\$18.2 billion economic gains in 2016. Globally, 82% of the total crop area for soybeans, 68% for cotton, 30% for maize, and 25% for oilseed rape were planted with GM varieties in 2014. Despite high adoption rates by farmers, the cumbersome regulatory processes and delayed cultivation approval procedures have reduced the value of innovation "the GM crops".

Quick action may be needed for strict legislation to distinguish between GE and transgenic crop. Modern breeding technologies, particularly GE, are highly feasible alternative options to GM crops and involve a reduced degree of regulatory oversight. New discoveries in GE technology and continuous progress in delivery systems that do not need to insert any specific foreign DNA in host cells for crop improvement may strongly challenge the legislative laws regulating the transgenic crops [130].

Traditional plant breeding approaches mainly depend on chromosomal modification via homologous recombination. Selection and crossbreeding have been applied for many years to screen the best performing varieties. Conventional plant breeding techniques have been used for several decades to detect novel traits and introduce them into individual plants for desired results. Traditional breeding has successfully developed many new cultivars, but these approaches need rigorous and continuous selection for many generations [256]. Genetic variability has significantly decreased due to the progressive evolution of many major crops through traditional breeding [257]. Therefore, modern plant breeding approaches have become essential to overcome the certain limitations of traditional breeding such as self-incompatibility, long generation time, heterozygosity, polyploidy, and time consuming.

Mutation breeding and engineering of transgenic plants are other crucial strategies used for crop improvement [258]. Conventional mutagenesis has helped to produced genetic variations that ultimately help in improving food quality and crop yield. For genetic analysis, natural or artificial mutagenesis has been induced using chemical and physical mutagens like ethyl methanesulfonate (EMS) and gamma rays [259]. Screening of large numbers of mutants is the biggest challenge. Such laborious, time-consuming, and untargeted breeding platforms cannot maintain pace with global food demands [258]. Foreign genes have been transferred into the elite crop lines to obtain desired traits through transgenic breeding. As compared to traditional breeding, transgenic techniques eliminate all crossing barriers and have increased genetic variability. Transgenic technology has provided enormous opportunities for crop improvement but, at the same time, provoked public concerns about its potential effect on human health and environment. Hence, commercialization of genetically modified crops (GMOs) is under strict control and limited by lengthy and expensive regulatory assessment procedures [260].

The advanced GE technology assists to produce precise and targeted mutations without the integration of any DNA sequence in plant genomes. This permits the development of non-transgenic crops with increased yield and improved quality and stress tolerance [261]. These approaches are speedy in contrast to traditional breeding techniques and allow development of transgene-free plants [156]. Plants produced via GE approaches are very similar to plants developed by conventional breeding. Additionally, GE technology takes less time to incorporate desired traits into the plant genome as compared to transgenic breeding, mutation breeding, and traditional breeding. It will take approximately 4–6 years to develop a GE plant with desired trait as compared to the transgenic breeding, which require 8–12 years for the creation of transgenic plant. On the other hand, mutation breeding and conventional breeding need 8–10 years approximately to obtain a desired phenotype [258]. The emergence of advanced GE tools not only revolutionized the world of science, but its economics

are also very spectacular as compared to genetically engineered plants. Generally, the total expenditure required to execute a single transformation event would have been approximately a quarter of a million US dollars [262], while the budget needed for GE could be \$30 [263], which is astonishingly economical and reliable. Consequently, large amounts of money can be saved on developing and approving genome-edited crops, avoiding laborious and time-consuming field experiments which normally demand many years to regulate a GM crop. In addition, it will eliminate the uncertainty and fear regarding the use of GM crops [212]

There is a primary need to review the current rules and regulations regarding GMOs. In addition, genome manipulation done via GE tools are quite different from a transgenic approach. For example, mutations produced by CIRSPR/Cas9 are small indels as compared to large gene sequence insertion or deletion [264]. Such small indels are most often produced in plants under normal growth environments and can also be generated through conventional mutagens. Additionally, in contrast to GMOs which need stable integration of foreign DNA in the genome, CRISPR/Cas9-mediated GE can be used to develop DNA-free non-transgenic plants with improved traits. As far as the regulatory affairs of these gene-edited plants are concerned, there is no international regulatory framework present at the moment. Two major stakeholders, the USA and European Union (EU), have opposite policies for the regulation of genome-edited plants. The United States Department of Agriculture (USDA) has exempted GE crops from its strict rules and regulations [265], while the EU holds the position to treat genome-edited plants as GMOs. Recently, the European Court of Justice has ordered the verdict that GE plants should be subjected to similar regulatory procedures as in the case of GMOs [266]. This judicial ruling may impede investment in GE techniques and limit their use in modern plant breeding platforms in European countries. In Germany, CIBUSTM canola cases are undecided and no legitimate information has been issued by the European Commission (EC) [267]. In 2011, independent legal experts at the EU suggested some legal categorization of modern plant breeding approaches, including GE technology. A committee was formed by the EC, called the "New Techniques Working Group", to evaluate plants developed using different breeding techniques that fall under the category of GMOs legislation. By the end of 2011, the assessment was completed and the report finalized but it was never published [268]. Although, some important regulatory entities of the EU (including the French High Council for Biotechnology, the European Plant Science Organization, German Academy of Sciences, and the British Biotechnology and Biological Sciences Research Council) have already proposed that the assessment of GE plants should be based on the specific trait improvement instead of technology executed to develop them. However, a study was carried out by the German Federal Agency for Nature Conservation about GE organisms and they decided that GE organisms must be treated under the same regulations as GMOs, arguing that since GE is a strategy to manipulate the genome to produce targeted modification linked with unfamiliar risks, regardless of genome alterations that happen in nature [269].

In several countries, emerging crop editing tools like meganulcease, TALENs, ZFNs, and CRISPR/Cas9 have been applied for the past decade and they do not come under the category of GMO regulatory laws. The USA and Canada regard gene editing as equivalent to traditional breeding. The United States Department of Agriculture (USDA) granted permission to regularize and develop the CRISPR/Cas9-mediated genome-edited crops. Besides the USA, many other countries such as Brazil, Chile, and Argentina have established advanced regulatory principles for genome-edited crops. Every new discovery in biotechnology has been flawlessly approved due to the trait-based scheme in Canada. A strong and authentic regulatory policy is required to distinguish between GMOs and GE plants. Unfortunately, many countries have not established a clear regulatory policy for GE plants. The extensive use of GE strategies brings many challenges for regulatory bodies, as it requires great technical expertise and reliable evaluating procedures for the regulation of GE crops. Evidently, science-based guidelines that judge genome edited plants in a similar way as plants developed by conventional breeding programs are required to boost the applications of GE for crop improvement. For this, many countries such as the United States, Argentina, Australia, Brazil, Canada, and Chile

have issued legal interpretations of various omissions in regulatory rules and exempted GE crops from the strict regulations of GMOs. However, this exemption may be dependent on some strict requirements like absence of foreign gene (Australia), no signs of pest characteristics (USA), and type of trait modification (Canada and other countries).

The USA is the main stakeholder in the world and several regulatory authorities govern the regulation of GE crops including the Food and Drug Administration (FDA), Environmental Protection Agency (EPA), Animal and Plant Health Inspection Service (APHIS), and the and United States Department of Agriculture (USDA). The current policy of the USA regarding GE crops was developed by the USDA and depends on the "Plant Protection Act". Any GE plant that poses pest characteristics and food safety issues is closely assessed and monitored by the regulatory bodies (USDA, EPA, and FDA). The USDA does not treat GE plants under GMOs regulations (https://www.aphis.usda.gov/ aphis/ourfocus/biotechnology/brs-news-and-information/pbi-details). The APHIS has proposed many verdicts regarding the risk assessment of DNA-free GE crops and suggested modifications to the rules in order to eradicate the legislation application about pest and GE crops in 2017 [270]. The United States Department of Agriculture (USDA) acknowledged gene editing as a much faster form of traditional breeding. The USDA has allowed more than ten case-by-case studies of genome editing for cultivation without regulatory permits. These included the development of a high level of amylopectin producing Wax corn by applying the CRISPR/Cas9 tool which has been mutated for the *Wx1* gene. Similarly, a CRISPR/Cas9 strategy was applied to produce browning resistance to white button mushrooms by mutating the *polyphenol oxidase* gene at the Pennsylvania State University [265]. Herbicide-resistant rape seed was produced by the RTDS mechanism. In soybean, drought-resistant genes such as *Drb2b* and *Drb2a* were knocked out using the CRIPSR/Cas9 system. *Setaria viridis* was subjected to the CRISPR/Cas9 technique for delayed flowering by disrupting the *ID1* gene and the *Camelina* genome was edited by Yield10 Bioscience for enhanced oil production. In addition, low phytate level corn has been established using Dow's ZFN, and resistant wheat against powdery mildew, soybean with a mutated *FAD3* gene, and potato with *PPO* knockout using a TALENs approach were also approved.

Similarly, there is no difference in Canada's approach towards the GE techniques from the techniques that have foreshadowed it. Canadian plants with novel traits (PNTs) regulations are activated only if the technique produces any specific trait, causing toxicity, allergenicity, and effects on any other organism. All the plant cultivars with specific traits are subjected to PNT regulations, irrespective of how they were produced, suggesting that the plant cultivar could be produced via conventional breeding, conventional mutagenesis, genetic engineering or gene editing. It is anticipated that some of GE techniques may produce novel cultivars that are PNTs, while many of them may not be treated under the PNTs regulations. Thus, in Canada, plant cultivars that are carried through the PNT regulations need open release approval from Health Canada and the Canadian Food Inspection Agency (CFIA) in order to register as approved cultivars for commercial use by industry [271].

Argentina established a functional regulatory framework for regularization of modern plant breeding products (Whelan 2015) [272]. Policy-makers and regulatory bodies have made flexible assessment protocols that depend on case-by-case evaluations. Fundamentally, the regulatory framework of Argentina determines the overall process of developing a GE plant. The plant developed without any transgene integration has been designated as non-GMO. Moreover, if any transgene strategy was applied but the final product is DNA-free, then this is also treated as non-GMO. A regulating body in Argentina, CONABIA, assesses the genome-edited material before giving approval. Under the rule No. 763/11, a simple deletion in genome is not regarded as a GM crop.

Most of the GE tools are introduced by industries, but the CRISPR/Cas technology was discovered by academic research groups. These academic institutions and different companies are contesting to establish intellectual property (IP) sets for speedy commercialization of CRISPR/Cas-based products. Since 2005, a 15 fold increase has been reported in the number of patent applications and 42 patent applications were registered in the USA in 2014. Over the last couple of years, investment in GE bio-enterprise has increased fivefold [273]. It was estimated that the market value of GE technology

was about \$1.84 billion at the end of 2014, and it is predicted to grow with a 13.75% compound annual growth rate of about \$3.51 billion by 2019 [274]. Additionally, the private companies that use CRISPR/Cas in the sector of agriculture, health, and industry have equally played a significant part in the current growth of the GE market. Over \$600 million has been received by major companies which use CRISPR/Cas technology over the last decade [275].

To conclude, regulatory authorities need to develop comprehensive regulatory frameworks which direct the utilization of GE tools without constraining research. Furthermore, issues of IP rights and licensing policies need to be scrutinized for GE plants which can be used for commercial purposes.

#### **8. Conclusion and Outlook**

The production of safe, low-cost, and nutritive food by adopting sustainable agricultural practices will be a huge task. In this regard, the availability of modern technologies to improve cultivars will be a vital aspect. GE is a powerful tool which is expected to play a crucial role in meeting the increasing demands of crop production to fulfill the needs of an exploding population under a climate change scenario. As compared to conventional breeding methodologies, the molecular breeding strategies aided by GE tools allow scientists to precisely target and edit for desired traits. GE can be used to enhance crop productivity, nutritional value, and develop resistance against biotic as well as abiotic stressors by improving the crop genome. The advanced tools in plant GE have been extensively employed to edit crops for a specific agronomic trait and have been utilized in several breeding platforms for carrying the desired trait for the development of an elite local variety. Thus, modern plant breeding approaches will increase the performance of plant breeding, and gene-edited elite cultivars can be approved for cultivation in specified locations without strict regulatory laws. For the last two decades SSNs such as meganucleases, ZFNs, and TALENs have revolutionized plant GE. These SSNs have many applications in plant GE and can be used for gene insertion, gene deletion, and increasing the efficiency of homologous recombination which allows for more precise and accurate events of gene replacement.

Beside other GE techniques, CRISPR/Cas9 is the most powerful tool for crop improvement. In many plant systems it has been vigorously applied over the last five years for combating abiotic and biotic stressors and to improve other agronomic traits. CRISPR/Cas9 as a GE technology for site-direct mutagenesis has many excellent characteristics including great target specificity, easiness to execute, and low cost, which are unachievable through conventional mutagenic strategies. Further, CRISPR/Cas9 is superior to other first-generation SSNs because RNAs guide the Cas9 nuclease instead of proteins. Several Cas9-mediated techniques are being employed in different plant varieties, and these techniques will offer exceptional knowledge about plant biology and facilitate us to develop improved cultivars with great accuracy and speed via modern plant breeding. Recently some striking developments have been achieved in the CRISPR/Cas9 toolbox to increase the targeted mutagenesis with increased efficiency via base editing, multiplex GE [276], and generation of DNA-free plants. The CRSIPR/Cas9 is a versatile tool for plant GE, due to the fact of its sophisticated toolbox of Cas9 variants such as the CRISPR/Cpf1 system and online accessible bioinformatics tools for designing highly precise delivery systems. The CRISPR/Cas9-based precise GE produces gene replacement, gene insertion, and knockout mutations that are rapidly being used to increase yield, improve quality, and enhance tolerance in crops to boost crop domestication and hybrid breeding. Moreover, CRISPR/Cas9 technology is gaining interest day by day and will be a fundamental GE approach to developing improved plants with desired traits that will aid in accomplishing the goal of zero hunger in the world.

Although CRISPR/Cas9-mediated GE has gained remarkable achievements in crop improvement, there are certain challenges that need to be addressed to develop a more efficient system for plant GE. This includes assembling pangenomes for crop improvement, programmed identification of candidate sites for gene editing via functional genomics, designing of highly efficient delivery systems for GE, and reducing the frequency of off-target editing, deciphering novel pathways for this reduction, and optimization of the Cas9 function. The major pitfalls of CRISPR/Cas9 is the inefficient delivery

system for plant transformation because the current protocols are limited to certain tissues [277], genotypes, and crop varieties. The packaging of Cas proteins into delivery vectors poses large barriers for efficient delivery of CRISPR/Cas machinery. Recently, some novel cargo-vector systems have been introduced which show promising potential for efficient delivery systems. For example, carbon nanotubes have been utilized to transfer CRISPR/Cas9 editing constructs into plant leaves. Some other nano-products such as mesoporous silica nanoparticles and layered double hydroxides also have great potential to broaden the accessibility of delivery systems, as they have high transformation efficiencies and little toxicity and cellular damage. Developing improved delivery systems will be vital for efficient targeted and more precise GE for crop improvement. On the other hand, the frequency of off-target effects needs to be addressed more comprehensively, as there are many safety issues linked with CRISRP/Cas9-based bio-products. Luckily, off-target mutations are mostly bearable in plants and mutants, and off-target effects can be detected and eliminated through segregation over successive crosses. Selection of Cas9 requiring long PAMs and designing of sgRNA with close affinity for target sequence, may help to reduce the off-target effects in the future. Therefore, continuous efforts are required to overcome these hurdles in order to increase the experimental versatility and applied skills of the CRISPR/Cas9 toolbox in the future.

The advancement in modern breeding approaches has been greatly acknowledged as an innovation in our capacity to manipulate genomes and has subsequently challenged our understanding and assessment of current regulatory policies. As GE tools are extensively employed in plants, the safety of GE plants is the matter of debate around the globe. Development of regulatory policies for novel crop innovations should be multidimensional, transparent, and be able to distinguish between GMOs and GE events. Hence, to explore the large prospective of modern plant breeding approaches for improved yield and food security, it is necessary to illuminate the clear status of these approaches, including GE, and to fix current regulatory uncertainties. Harnessing the innovative ideas of system biology, synthetic biology, next-generation sequencing, and the latest developments in functional genomic approaches integrated with the advanced tools of CRISPR/Cas9 will permit the development of smart crops with higher yields and improved qualities. In the near future, CRISPR/Cas9 technology can be integrated with speed breeding programs to revolutionize the global agriculture and promise of food security.

**Author Contributions:** A.R. conceived the idea. A.R. and M.K. wrote the manuscript. F.S. helped in original draft preparation. F.A.J. and A.R. developed figures and organized tables. M.K.H. helped in the literature review. F.A.J., S.Y., F.S., G.M., and H.M.I.A. read, reviewed, and edited the manuscript. M.S.K. provided expert opinion, technical assistance, and finalized the manuscript. All authors listed have made substantial, direct, and intellectual contributions to the work and approved the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We are grateful to all the researchers whose contributions have been cited in this review paper, which have helped us to prepare this review paper. Furthermore, we apologize to those authors whose excellent work could not be cited due to space limitations.

**Conflicts of Interest:** The authors declare no conflict of interest.
