Rice blast is one of the most destructive diseases affecting rice worldwide. The adoption of host resistance has proven to be the most economical and effective approach to control rice blast. In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be powerful tools for the improvement of crops via gene-specific genome editing, and CRISPR/Cas9 is thought to be the most effective SSN. Here, we report the improvement of rice blast resistance by engineering a CRISPR/Cas9 SSN (C-ERF922) targeting the OsERF922 gene in rice. Twenty-one C-ERF922-induced mutant plants (42.0%) were identified from 50 T₀ transgenic plants. Sanger sequencing revealed that these plants harbored various insertion or deletion (InDel) mutations at the target site. We showed that all of the C-ERF922-induced allele mutations were transmitted to subsequent generations. Mutant plants harboring the desired gene modification but not containing the transferred DNA were obtained by segregation in the T₁ and T₂ generations. Six T₂ homozygous mutant lines were further examined for a blast resistance phenotype and agronomic traits, such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight. The results revealed that the number of blast lesions formed following pathogen infection was significantly decreased in all 6 mutant lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T₂ mutant lines and the wild-type plants with regard to the agronomic traits tested. We also simultaneously targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs (C-ERF922S1S2 and C-ERF922S1S2S3) to obtain plants harboring mutations at two or three sites. Our results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice.

Summary: Targeted mutagenesis via genome‐editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms often determine important agronomic traits of crops. Genome‐editing‐based single‐base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome‐editing technologies, base editing has emerged as a novel and efficient genome‐editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR–Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base‐editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted.

Rice (Oryza sativa L.) is the major food source for more than three billion people of the world. In the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties. However, stagnation in rice yield has been reported in recent decade owing to several factors including the emergence of pests and phyto pathogens, climate change, and other environmental issues posing great threat to global food security. There is an urgent need to produce more rice and associated cereals to satisfy the mammoth task of feeding a still growing population expected to reach 9.7 billion by 2050. Advances in genomics and emergence of multiple genome-editing technologies through use of engineered site-specific nucleases (SSNs) have revolutionized the field of plant science and agriculture. Among them, the CRISPR/Cas9 system is the most advanced and widely accepted because of its simplicity, robustness, and high efficiency. The availability of huge genomic resources together with a small genome size makes rice more suitable and feasible for genetic manipulation. As such, rice has been increasingly used to test the efficiency of different types of genome editing technologies to study the functions of various genes and demonstrate their potential in genetic improvement. Recently developed approaches including CRISPR/Cpf1 system and base editors have evolved as more efficient and accurate genome editing tools which might accelerate the pace of crop improvement. In the present review, we focus on the genome editing strategies for rice improvement, thereby highlighting the applications and advancements of CRISPR/Cas9 system. This review also sheds light on the role of CRISPR/Cpf1 and base editors in the field of genome editing highlighting major challenges and future implications of these tools in rice improvement.

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

Bacterial blight (BB) and bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, respectively, are two devastating diseases in rice planting areas worldwide. It has been proven that adoption of rice resistance is the most effective, economic, and environment-friendly strategy to avoid yield loss caused by BB and BLS. As a model system for plant—pathogen interaction, the rice—X. oryzae pathosystem has been intensively investigated in the past decade. Abundant studies have shown that the resistance and susceptibility of rice to X. oryzae is determined by molecular interactions between rice genes or their products and various pathogen effectors. In this review, we briefly overviewed the literature regarding the diverse interactions, focusing on recent advances in uncovering mechanisms of rice resistance and X. oryzae virulence. Our analysis and discussions will not only be helpful for getting a better understanding of coevolution of the rice innate immunity and X. oryzae virulence, but it will also provide new insights for application of plant R genes in crop breeding.