The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 system (Cas9) has been used at length to optimize multiple aspects of germplasm resources. However, large-scale genomic research has indicated that novel variations in crop plants are attributed to single-nucleotide polymorphisms (SNPs). Therefore, substituting single bases into a plant genome may produce desirable traits. Gene editing by CRISPR/Cas9 techniques frequently results in insertions–deletions (indels). Base editing allows precise single-nucleotide changes in the genome in the absence of double-strand breaks (DSBs) and donor repair templates (DRTs). Therefore, BEs have provided a new way of thinking about genome editing, and base editing techniques are currently being utilized to edit the genomes of many different organisms. As traditional breeding techniques and modern molecular breeding technologies complement each other, various genome editing technologies have emerged. How to realize the greater potential of BE applications is the question we need to consider. Here, we explain various base editings such as CBEs, ABEs, and CGBEs. In addition, the latest applications of base editing technologies in agriculture are summarized, including crop yield, quality, disease, and herbicide resistance. Finally, the challenges and future prospects of base editing technologies are presented. The aim is to provide a comprehensive overview of the application of BE in crop breeding to further improve BE and make the most of its value.

Background: High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. Results: We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T₀ generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T₀ generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. Conclusions: This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

Rice (Oryza sativa L.) feeds half the world’s population and serves as one of the most vital staple food crops globally. The brown planthopper (BPH, Nilaparvata lugens Stål), a major piercing–sucking herbivore specific to rice, accounts for large yield losses annually in rice-growing areas. Developing rice varieties with host resistance has been acknowledged as the most effective and economical approach for BPH control. Accordingly, the foremost step is to identify BPH resistance genes and elucidate the resistance mechanism of rice. More than 70 BPH resistance genes/QTLs with wide distributions on nine chromosomes have been identified from rice and wild relatives. Among them, 17 BPH resistance genes were successfully cloned and principally encoded coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) protein and lectin receptor kinase (LecRK), as well as proteins containing a B3 DNA-binding domain, leucine-rich repeat domain (LRD) and short consensus repeat (SCR) domain. Multiple mechanisms contribute to rice resistance against BPH attack, including transcription factors, physical barriers, phytohormones, defense metabolites and exocytosis pathways. Plant hormones, including jasmonic acid (JA), salicylic acid (SA), ethylene (ET), abscisic acid (ABA), gibberellins (GAs), cytokinins (CKs), brassinosteroids (BRs) and indoleacetic-3-acid (IAA), play crucial roles in coordinating rice defense responses to the BPH. Here, we summarize some recent advances in the genetic mapping, cloning and biochemical mechanisms of BPH resistance genes. We also review the latest studies on our understanding of the function and crosstalk of phytohormones in the rice immune network against BPHs. Further directions for rice BPH resistance studies and management are also proposed.

Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T₀ transgenic plants. A proof-of-concept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms.

IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the