Application of CRISPR Technology in Plant Improvement: An Update Review

The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) method is a versatile technique that can be applied in crop refinement. Currently, the main reasons for declining agricultural yield are global warming, low rainfall, biotic and abiotic stresses, in addition to soil fertility issues caused by the use of harmful chemicals as fertilizers/additives. The declining yields can lead to inadequate supply of nutritional food as per global demand. Grains and horticultural crops including fruits, vegetables, and ornamental plants are crucial in sustaining human life. Genomic editing using CRISPR/Cas9 and nanotechnology has numerous advantages in crop development. Improving crop production using transgenic-free CRISPR/Cas9 technology and produced fertilizers, pesticides, and boosters for plants by adopting nanotechnology-based protocols can essentially overcome the universal food scarcity. This review briefly gives an overview on the potential applications of CRISPR/Cas9 and nanotechnology-based methods in developing the cultivation of major agricultural crops. In addition, the limitations and major challenges of genome editing in grains, vegetables, and fruits have been discussed in detail by emphasizing its applications in crop refinement strategy.

Prokaryotic type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein9) adaptive immune system has been applying in many plants for editing target gene(s). The CRISPR/Cas9 technology is superior to other programmable nucleases, such as ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), for its fast activating, simplicity, high efficiency, and economically feasibility. Thus, the current study have mainly focused on the study of CRISPR/Cas9 technology against plant biotic agents like fungus, bacteria, virus, and insect pests. Though this technology is highly applicable against all biotic agents but the success rate based on the previously published papers clarified that it is more effective towards to viruses and fungi. This technology may be used in editing both the target sites either of plants or biotic agents. In both the cases it produces desirable modification in the target sites, makes this technology more applicable to produce biotic agent resistant crop variety. So this editing technology is considered not only a powerful tool for genome editing in both plant and biotic agent but also for developing non-transgenic biotic stress resistant crop variety. The current review may explore the role of CRISPR/Cas9 technology in introgression of QTL, deletion of unwanted genes for crop plants resistant against specific pests and environmental stresses.

Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.

CRISPR/Cas9 revolutionizes Brassica crop improvement by enhancing yield, quality, and stress resistance, providing a precise and versatile tool for genetic and agronomic advancements. The rapidly advancing CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technologies are being employed in both diploid and polyploid species of Brassica for gene functions and precise genetic improvements. CRISPR/Cas technology has sparked significant attention among the scientific community due to its affordability, precision, and effectiveness compared to other genome editing techniques. The recent discoveries highlight the diverse applications of the CRISPR/Cas9 genome editing tool in enhancing agriculturally important traits in Brassica species. This technology has been utilized to improve yield, quality, and resistance to both biotic and abiotic stresses globally. Here, we present an overview that encourages researchers to explore and improve the functionality and genetic progress of Brassica U-triangle species utilizing genome editing technologies. In addition, ethical considerations and concerns associated with CRISPR technologies are addressed, providing valuable insight into how CRISPR/Cas9 tools and have revolutionized crop improvement with special emphasis on Brassica for various agronomically and nutritionally important traits.

Plants are subjected to biotic and abiotic stresses regularly, which irreparably harm agricultural production. Eco-friendly and sustainable technology to deal with this challenge is to breed abiotic stress tolerant cultivars. To generate crop plants conferring resistance against stresses, conventional breeding was used in the past, but because of the complex heredity of abiotic stress tolerance traits, such techniques remain insufficient in making greater enhancement. Genome-engineering based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein9) has shown enormous potential in developing climate-resilient cultivars. Likewise, the development of chickpea transgenic lines by knockout of 4CL and REV7 genes exhibits drought tolerance which establishes a foundation for future studies in chickpea. In addition, the CRISPR-Cas9 system can boost yield potential under abiotic stress situations by producing non-transgenic plants having the required characteristics. This review article discusses the validation of gene function based on the CRISPR-Cas9 for the development of abiotic stress-tolerant crop plants, emphasizing the chickpea to open the new ventures of generating abiotic stress-tolerant chickpea varieties.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system was initially identified in bacteria and archaea as a defense mechanism to confer immunity against phages. Later on, it was developed as a gene editing tool for both prokaryotic and eukaryotic cells including plant cells. CRISPR/Cas9 approach has wider applications in reverse genetics as well as in crop improvement. Various characters involved in enhancing economic value and crop sustainability against biotic/abiotic stresses can be targeted through this tool. Currently, CRISPR/Cas9 gene editing mechanism has been applied on around 20 crop species for improvement in several traits including yield enhancement and resistance against biotic and abiotic stresses. In the last five years, maximum genome editing research has been validated in rice, wheat, maize and soybean. Genes targeted in these plants has been involved in causing male sterility, conferring resistance against pathogens or having certain nutritional value. Current review summarizes various applications of CRISPR/Cas system and its future prospects in plant biotechnology targeting crop improvement with higher yield, disease tolerance and enhanced nutritional value.

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes (S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.

Several plant pathogens severely affect crop yield and quality, thereby threatening global food security. In order to cope with this challenge, genetic improvement of plant disease resistance is required for sustainable agricultural production, for which conventional breeding is unlikely to do enough. Luckily, genome editing systems that particularly clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has revolutionized crop improvement by enabling robust and precise targeted genome modifications. It paves the way towards new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. In this review, the challenges, limitations and prospects for conventional breeding and the applications of CRISPR/Cas9 system for the development of transgene-free disease-resistant crops are discussed.

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system has been regularly applied for genome editing and gene function identification in wild soybean (Glycine max) cultivars. However, till date no studies have demonstrated successful mutagenesis in wild soybean (Glycine soja) which is the ancestor of Glycine max and rich in stress tolerance genes. In the current study, we report the successful creation of mutations in the loci encoding plasma membrane Na+/H+ antiporter (SOS1) and nonselective cation channels (NSCC) in wild soybean hairy roots using the CRISPR/Cas9 system. Two genes, GsSOS1 and GsNSCC, were mutagenized with frequencies of 28.5% and 39.9%, respectively. Biallelic mutations in GsSOS1 were detected in transgenic hairy roots. GsSOS1 mutants exhibited altered Na+/K+ ratios in the roots under both control and salt-treated conditions. However, no significant effects of GsNSCC mutation on Na+/K+ ratios were observed. RNA-Seq analysis revealed that both GsSOS1 and GsNSCC mutation altered the transcription profiles in mutant roots. Many differentially expressed gene sets that are associated with various cellular functions were identified. Our results demonstrated that CRISPR/Cas9 systems as powerful tools for wild soybean genome editing and would significantly advance the gene mining and functional identification in wild soybean.

Soil salinity is a major threat to global agriculture, limiting plant growth and lowering crop yields. Recent advances in CRISPR/Cas9 genome editing technology provide unprecedented precision and efficiency for addressing these challenges by directly modifying the central dogma (CD) of molecular biology in plants. The CD naturally lends itself to tighter multi-level regulation, where transcription and translation are both under control at the same time. A multilayer component of CD such as epigenetic modification, transcription, post-transcriptional modification, translation, and post-translational modification contributes significantly to stress tolerance. Strict control of CD components by Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associated protein 9 (CRISPR/Cas9) might lead to the generation of climate smart crops. This review delves into the latest developments in the CRISPR toolbox that improve plant salt tolerance. By targeting key genes involved in transcription and translation, CRISPR/Cas9 makes it easier to modify critical components of the central dogma, allowing plants to better manage salt stress. We explore various CRISPR-based strategies, including base editing, prime editing, transcription regulation, multiplexing, RNA and many more, that reprogram gene expression and protein function to improve salt tolerance. In addition, we discuss how CRISPR can be combined with transcriptional regulation and epigenetic modifications to provide a comprehensive approach to salinity resistance for plants. The review also addresses the issues of off-target effects and efficient delivery systems, recommending novel solutions to improve the precision and applicability of CRISPR technology. This review emphasizes the transformative potential of CRISPR in modifying the central dogma to develop salt-tolerant crops, thereby contributing to sustainable agriculture and global food security.

Drought stress is one of the most considerable threats to global agricultural food security, causing yield losses worldwide. Therefore, the search for effective genetic and molecular methods for developing cultivars that are tolerant or resistant to harsh environments has been more intensive over the last decades. Apart from time-consuming conventional breeding techniques, biotechnologists are now investigating modern genome editing tools for engineering tolerance and resistance to various biotic and abiotic stresses in crops. Various genetic engineering techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were developed based on the discovery of the DNA structure. However, these methods have limitations, with ZFNs being prone to errors due to their limited base pair recognition, and TALENs requiring a complex protein engineering process and struggling to cleave methylated DNA. In recent years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and its alternatives have gained popularity in plant biotechnology. Out of the genome editing techniques mentioned earlier, CRISPR/Cas9 is becoming more popular because it's faster and easier to use. Given that drought is now a significant threat to global agriculture due to the drying of arable lands, this review focuses on how we can use CRISPR genome editing to enhance crop tolerance to drought stress and explores its future potential.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

CRISPR-Cas genome editing system: A versatile tool for developing disease resistant crops

Genetic toolsets are essential for gene discovery, elucidating biological pathways, and accelerating molecular breeding of superior crops in plant biology and agriculture. Among these, the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system has emerged as a powerful and indispensable tool for precise genome editing in maize (Zea mays L.). This protocol presents a comprehensive, maize-specific approach to constructing CRISPR vectors and analyzing transgenic plants carrying targeted gene mutations. It is organized into two major sections. The first section provides a step-by-step guide for designing guide RNAs and oligonucleotides (oligos) to construct CRISPR vectors containing one, two, four, or multiplexed (up to eight) single-guide RNAs (sgRNAs). It also describes the modular assembly of these sgRNAs with the Cas9 expression cassette using the Gateway cloning strategy to streamline vector construction. The second section focuses on genotyping CRISPR-edited plants by detecting and characterizing target mutations. Four complementary methods are outlined: (1) the T7 endonuclease I (T7EI) assay, (2) restriction enzyme digestion, (3) Sanger sequencing of PCR amplicons, and (4) high-throughput sequencing. Methods 1 and 2 offer rapid and cost-effective screening for small insertions or deletions (indels), while methods 3 and 4 provide high-resolution and scalable mutation analysis. Together, this workflow offers researchers an efficient, flexible, and reliable system for genome editing and mutation validation in maize, supporting both functional genomics studies and trait improvement applications.