CRISPR/Cas Technology Revolutionizes Crop Breeding

Crop breeding is an important global strategy to meet sustainable food demand. CRISPR/Cas is a most promising gene-editing technology for rapid and precise generation of novel germplasm and promoting the development of a series of new breeding techniques, which will certainly lead to the transformation of agricultural innovation. In this review, we summarize recent advances of CRISPR/Cas technology in gene function analyses and the generation of new germplasms with increased yield, improved product quality, and enhanced resistance to biotic and abiotic stress. We highlight their applications and breakthroughs in agriculture, including crop de novo domestication, decoupling the gene pleiotropy tradeoff, crop hybrid seed conventional production, hybrid rice asexual reproduction, and double haploid breeding; the continuous development and application of these technologies will undoubtedly usher in a new era for crop breeding. Moreover, the challenges and development of CRISPR/Cas technology in crops are also discussed.


Introduction
In the future, agricultural production faces major challenges from a rapidly increasing human population and severe environmental stresses.Crop yield is a complex quantitative trait governed by many genes and environment factors, and some key genes and agronomic traits have gradually weakened or been lost during crop domestication [1].Traditional breeding methods, such as crossing breeding and mutation breeding, have achieved increasing crop yield, but they also have many limitations in breeding superior varieties due to the lack of valuable natural germplasms, the obstacles of undesired genome incorporation or linkage drag, and their time consuming and laborious screening process [2].Compared with traditional methods, biotechnologies including the use of transgene, gene editing, double haploid technique, and synthetic apomixis provide new opportunities for crop breeding [2][3][4][5][6].Transgenic technology by ectopically expressing specific genes overcomes the limitations associated with conventional techniques and has played an important role in the genetic improvement of crop yield, quality, and resilience towards biotic and abiotic stresses; however, its use has been seriously restricted due to the time-consuming process, risk assessment, and regulatory process [7].As an important technique, doubled haploid technology can effectively accelerate the development of new crop varieties and shorten the breeding period by several years through the directly generating homozygous plants, which have been widely used in multiple major crops such as maize, wheat, rice, and soybean.The combination of double haploid breeding with traditional breeding methods, molecular marker-assisted selection, and especially gene editing technology will greatly improve the efficiency of crop breeding [8,9].improvements [28], providing a feasible tool to identify key genes and cultivate desired maize lines.
Soybean is an important oil-bearing crop, whose oils are primarily composed of polyunsaturated fatty acids, monounsaturated fatty acids, and saturated fatty acids.Comparatively, the high content of monounsaturated fatty acids in vegetable oils is beneficial for human health and food shelf-life [46].Recently, an elevated monounsaturated fatty acids soybean germplasm was created by knocking-out GmPDCTs, providing a feasible strategy for the improvement of soybean nutrition and shelf stability [46].Soybean root rot disease is a most destructive disease majorly caused by Phytophthora sojae (P.sojae) that endangers the entire growth cycle of soybean and seriously affects its yield and quality [47].Recently, an excellent germplasm with high resistance to several P. sojae strains was engineered by editing GmTAP1 [47].Moreover, key genes associated with yellow-green variegation of leaf [48], plant architecture [49], and fatty acid anabolic metabolism [50] have been well characterized in soybean.

Ushering in a New Era of Crop De Novo Domestication
For a long time, crop domestication has mainly focused on selecting desirable traits related to yield, leading to the gradual loss of potentially useful traits in wild species such as pest and disease resistance, abiotic stress tolerance, and nutritional quality.Traditional wild crop domestication methods are still an option to create new germplasms, but they are time-consuming, labor-intensive, and difficult to aggregate multiple traits [51,52].In 2017, a strategy for wild species de novo domestication was proposed using genome editing techniques [52].In particular, CRISPR/Cas technology has achieved rapid wild crop de novo domestication by simultaneously editing key genes controlling agronomic traits, showing attractive prospects for effectively developing elite varieties [51][52][53][54][55][56][57].
In tomato, alleles conferring desirable traits were discovered in wild Solanum pimpinellifolium.These allelic changes were engineered by editing six important loci essential for yield and nutritional value in modern tomatoes.The engineered lines had significant increases in fruit size, fruit number, and fruit lycopene [58].Novel germplasms with enhanced productivity were created in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) by editing orthologues of modern tomato genes controlling plant architecture and yield-related traits, realizing the rapid creation of elite genomic resources in distantly related orphan crops [59].Stress-tolerant wild-tomato species with desirable traits were engineered using a multiplex CRISPR/Cas9 technology, which had domesticated phenotypes and retained parental stress-tolerant traits [60].Moreover, a 'two-in-one' strategy for stress-tolerant and multi-scenario cultivation breeding in tomatoes was devised through combining wild species de novo domestication with CRISPR/Cas, generating male-sterility in modern cultivars [61].Recently, rapid de novo domestication of wild allotetraploid rice was realized by editing O. alta homologs of the genes controlling various agronomic traits in diploid rice, providing an effective way to breed new varieties aggregating desired traits via wild rice de novo domestication [51,62].These studies demonstrate that CRISPR/Cas technology is a powerful tool for wild species de novo domestication to create new desirable varieties, ushering in a new era of crop breeding to utilize wild species genetic diversity in major and orphan crops.

Breaking Breeding Bottlenecks of Tradeoff Effects
Due to linkage drags or gene pleiotropy, crop breeding is often subject to complex tradeoffs between traits, such as high yield and stress/disease resistance, yield and nutritional quality, and yield and plant architecture.In particular, the tradeoff effects caused by gene pleiotropy have become the bottleneck of multi-traits pyramiding breeding [63][64][65][66].Recently, CRISPR/Cas-mediated editing of cis-regulatory regions was used in different crops to generate novel beneficial alleles with improved stress resistance, yield, and quality [67,68].Unlike editing a coding region, editing a cis-regulatory region can fine-tune the expression level or profile of the target gene without disrupting its function, thereby optimizing the tradeoff effects of the pleiotropic gene [67,69].Recently, various tradeoff effects have been subtly solved in rice, maize, and wheat using CRISPR/Cas technology.
In rice, OsSWEET14 has tradeoff effects on disease resistance with plant height, tiller number, and seed size, whose loss-of-function mutation confers rice with enhanced disease resistance, but leads to small seed and delayed growth [70,71].Using CRISPR/Cas technology, a broad-spectrum resistant rice with normal tiller number and seed size was developed by editing the TALE-binding elements in OsSWEET11 and OsSWEET14 promoters [72].IPA1, a pleiotropic gene regulating various agronomic traits and stress resistance, has tradeoff effects on rice yield-related traits such as panicle size and tiller number [73][74][75][76][77][78][79][80][81][82].New rice germplasms with enhanced yield were created by deleting a key cis-regulatory region controlling IPA1 expression pattern, which subtly decoupled its tradeoff effect on grains per panicle and tiller number [66,83].SLG7 is a key gene regulating grain slenderness and low chalkiness.By editing the AC II element-containing region in the promoter, CRISPR/Cas-edited SLG7 alleles with increased expression levels exhibited better appearance quality without affecting yield and eating quality [84].Similarly, in hybrid rice, editing the regulatory regions of HEI10 led to an altered expression level and genetic recombination, which may be used for developing elite varieties [85].Recently, a CRISPR/Cas12a promoter editing (CAPE) system has been developed to improve rice agronomic traits by editing specific gene promoters.A high yield rice with better lodging resistance than Green Revolution OsSD1 mutant was generated by editing the OsD18 promoter [86].Moreover, a high-efficiency prime-editing system was used to create resistant alleles with broadspectrum resistance by knocking-in TAL effector binding elements from OsSWEET14 into the promoter of dysfunctional xa23 [87].These research examples provide alternative strategies for the creation of quantitative variations of agronomic traits.
In maize, although the key components of the CLAVATA-WUSCHEL signal pathway impact yield formation due to their tradeoff effects on ear meristem activity and ear development, their weak alleles show few yield effects [88][89][90][91][92][93].Recently, elite weak alleles with increased grains per ear and maize yield were created by editing the key regulatory regions of ZmCLE7 and ZmFCP1 [94].Since the CLAVATA-WUSCHEL signal pathway that coordinates stem cell proliferation with differentiation is conserved in diverse higher plant species, this study provides an alternative idea to improve yield traits of other crops.
In wheat, Mildew resistance locus O (MLO), a pleiotropic susceptibility gene, has tradeoff effects on disease resistance and yield-related traits [44,95,96].Using CRISPR/Cas technology, a mlo resistance allele (Tamlo-R32) with normal growth and yield was engineered in multiple varieties by deleting a large fragment in the MLO-B1 locus, which led to ectopic activation of TaTMT3B and thereby rescued growth and yield penalties caused by MLO disruption [44].These studies provide effective strategies for developing high-yielding crop varieties with stress resistance by decoupling the tradeoff on different traits caused by gene pleiotropy.

Accelerating Conventional Production of Crop Hybrid Seed
Heterosis is a breakthrough in crop breeding which has greatly improved crop yield.However, since offspring cannot maintain their heterosis due to genetic separation of traits, it is a time-consuming, laborious, and costly process to produce hybrid seeds every year [97,98].The wide use of male-sterile lines has greatly enhanced hybrid seed yield and quality in crop breeding.Recently, CRISPR/Cas technology has shown its unique advantages to unravel the mechanism of male sterility and develop male-sterile lines [99].Many male-sterile-related genes have been well characterized in maize [100], rice [101][102][103][104][105], wheat [106], and soybean [107], and improved the understanding of the molecular mechanisms that control male sterility in crops.Importantly, increasing CRISPR-edited malesterile lines have been generated in maize by knocking out ZmMS26 or ms8 [108,109], rice by editing TMS5, OsOPR7, or CYP703A3 [99,[110][111][112][113][114][115], wheat by targeting TaNP1, Ms1, or Ms45 [116][117][118], foxtail millet [119], and soybean [120].Thus, the CRISPR/Cas technology has provided a powerful tool for the generation of male-sterile lines and will greatly promote commercial hybrid seed production in different crops.
In the production of hybrid rice seeds, the restorer lines must be removed before seed harvesting to avoid contaminating undesired inbred line seeds, which results in the labor-intensive and costly hybrid seed production.Thus, the use of female-sterile lines as pollen donors might be an effective strategy to solve this problem [98].Unlike male-sterility, thermo-sensitive female sterility has been rarely reported due to the lack of desired germplasms, but it is very important for crop hybrid seed production via full mechanization [121].Using CRISPR/Cas technology, the first thermo-sensitive female sterility gene, AGO7/TFS1, was identified to engineer a female sterility line without causing defects in vegetative or male reproductive development.As a restorer line, its field trial showed a high seed-setting rate of hybrid panicles, paving a new path for fully mechanized hybrid seed production like conventional rice [98,121].

Promoting Hybrid Rice Asexual Reproduction
Heterosis refers to the better performance of an F1 hybrid than both parent lines.However, this performance is easily lost due to the random segregation of genetic information in the offspring generations.Apomixis has the potential to allow offspring to retain valuable traits through asexual reproduction, which can lower seed production costs and is important for crop breeding, but apomixis is absent in major crops [122,123].Thus, how to generate apomixis in crops has become a cutting-edge research hotspot in the field of botany.Recently, genome editing-mediated apomixis technology has realized heterosis fixation in hybrid offspring.In rice, clonal progeny retaining parental heterozygosity was obtained by CRISPR-editing BABY BOOM1 (BBM1), BBM2, and BBM3, and its asexual-propagation traits can be stably inherited in multiple generations of clones [124].Similarly, by combining heterozygosity fixation with haploid induction by simultaneous editing of REC8, PAIR1, OSD1, and MTL, Wang et al. generated hybrid rice plants that could propagate clonally through seeds, realizing self-propagation and stable transmission of elite F1 hybrid crops [125,126].Excitingly, in hybrid rice, high-frequency synthetic apomixis was achieved by simultaneous editing of PAIR1, REC8, and OSD1, and clonal progeny could stably retain the phenotype and genotype of F1 hybrid in successive generations [127].These studies suggest that the emergence of synthetic apomicts generated by CRISPR/Cas technology will provide efficient ways to utilize F1 hybrid heterosis, which will hopefully promote the realization of converting hybrids to apomixis in a sustainable way.
Recently, CRISPR/Cas9 technology-mediated haploid induction systems have been developed in different crops.In maize, a haploid induction editing technology (HI-EDIT), a Haploid-Inducer Mediated Genome Editing (IMGE) system, an approach combining haploid induction with a robust haploid identification marker, and a CRISPR/dCas9mediated gene activation toolkit were established to effectively generate genome-edited haploids [145][146][147][148]. Using a CRISPR/Cas9 vector with an enhanced green fluorescent protein expression cassette, an efficient haploid induction system was developed by editing BnaDMP genes in Brassica napu [149].In foxtail millet, haploid induction has been achieved by CRISPR/Cas9-mediated mutation of SiMTL, providing a possible application of double haploid technology in its breeding [150].Importantly, a fast technique for visual screening of wheat haploids was developed by combining the haploid inducer generated by editing TaMTL and embryo-specific anthocyanin markers, providing a promising strategy for a large-scale haploid inducer in different crops [151].Recently, a RUBY reporter system, a background-independent and efficient marker for haploid identification, has been established, which enables easy and accurate haploid identification in maize and tomato, which will be promising in double haploid breeding in different crops [152].

Conclusions and Future Perspectives
The advent and updating of CRISPR/Cas technologies have paved the way for gene function analysis and crop breeding, providing unprecedented opportunities for the generation of novel genetic variation, rapid crop de novo domestication, creation of male-sterile lines and female-sterile lines, development of double haploid technology, and precise pyramiding breeding (Figure 1).In particular, the upgrade and integration of genome editing, haploid induction, and apomixis technologies will usher in a new era for crop breeding [125].Although many CRISPR-Cas-edited crop materials have been generated in different crops, only few have been approved for commercial production and are entering the market worldwide due to regulatory policies such as a CRISPR/Cas9 waxy corn [26], a CRISPR-edited GABA-enriched tomato [153].Currently, the United States, Japan, Brazil, Argentina, Israel, Canada, and Australia have adopted relatively loose regulatory policies on gene editing crops, that is, gene editing products that do not contain foreign genes are Although many CRISPR-Cas-edited crop materials have been generated in different crops, only few have been approved for commercial production and are entering the market worldwide due to regulatory policies such as a CRISPR/Cas9 waxy corn [26], a CRISPR-edited GABA-enriched tomato [153].Currently, the United States, Japan, Brazil, Argentina, Israel, Canada, and Australia have adopted relatively loose regulatory policies on gene editing crops, that is, gene editing products that do not contain foreign genes are exempt from regulation.It is expected that more gene-edited plant products will accelerate commercialization.In 2021, the United Kingdom set to loosen rules for gene-edited crops and animals whose genes have been edited with precision techniques such as CRISPR.It will speed research and stimulate investment in these fields (https://www.science.org/content/article/uk-set-loosen-rules-gene-edited-crops-and-animals.Accessed on 26 May 2021).In China, the Guidelines for the Safety Evaluation of Gene Editing Plants for Agricultural Use (Trial) were issued in 2022, and the first safety certificate for the application of CRISPR-Cas-edited soybean was approved in 2023 (http://www.moa.gov.cn/ztzl/zjyqwgz/spxx/202304/t20230428_6426465.htm,accessed on 28 April 2023).
Notably, many CRISPR/Cas products have only been tested for their characters under simulated conditions, and there is a lack of field trials to evaluate their final field performance, which seriously hinders their application in production [100].Thus, it is urgent to focus on field trials of CRISPR/Cas-edited crops and thus, promote their commercial production.For example, editing of KRN2 or OsKRN2 could significantly enhance maize and rice grain yield without apparent negative impacts on other agronomic traits in their field trials [20].The ZmRAVL1-KO line displayed greater field yields than wild-type plants under different planting densities in two locations, showing excellent field traits for high planting density [22].The higher yields and superior agronomies of 12 CRISPR/Cas waxy corn hybrids have been validated by field trials at 25 locations in the USA, and their precommercial production was launched in 2019 [26].Thus, field trials like these will inevitably promote the commercial production of new gene-edited crops.
Low efficiency and high genotype dependency on genetic transformation processes are the major bottlenecks limiting the widespread application of CRISPR/Cas technology in different crops and elite varieties [154,155].Recently, developed genotype-independent enhanced gene transformation systems, by overexpressing the morphogenic genes (TaWOX5 and Wus2/Bbm), could significantly increase genome-edited plant regeneration in wheat, rye, barley, maize, and rice, providing new ways to expand genetic transformation and genome editing across the Poaceae family [155,156].Further optimizing transformation methods will advance genome editing on a wider range of crop species and varieties.Moreover, it is imperative to develop CRISPR/Cas systems with higher editing efficiency, lower off-target activity, more editing ways, and wider editing range, which will make them more effective and flexible in crop breeding.Recently, an optimized Cas12a base editor (Cas12a-ABE) has been established to introduce inheritable multiplex base edits in wheat and maize, which will assist in optimizing genome editing systems in a wide range of crop species [157].With the continuous development of CRISPR/Cas technology and its deep integration with other breeding techniques, it will become a popular strategy for breeders to precisely generate novel germplasms in different crops and usher in a new era of crop breeding.

Plants 2023 , 15 Figure 1 .
Figure 1.The application of CRISPR/Cas technology in the generation of new germplasms, analysis of gene function, and the iterative upgrading of multiple breeding technologies in different crops.

Figure 1 .
Figure 1.The application of CRISPR/Cas technology in the generation of new germplasms, analysis of gene function, and the iterative upgrading of multiple breeding technologies in different crops.