CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.

As a staple food crop, Oryza sativa L. is not only the basis of global food and nutrition security but also an important cornerstone of national economic development and social stability. However, the growth of rice is often accompanied by the threat of rice blast, which can lead to the death of seedlings or plants before heading. In the later stages of growth, a severe leaf blast infection will reduce the leaf area at the filling stage, thereby reducing the grain yield. The study of rice blast resistance genes and susceptibility genes is a key strategy for controlling the occurrence of rice blast and ensuring sustainable rice production. This paper reviews the impact of rice blast on the global economy and food security in recent years, describes the immune mechanism of rice blast resistance, and introduces the latest progress in related research. At the same time, the main genes of rice blast resistance and the resistance-related genes, as well as the susceptibility genes identified or cloned in recent years, are summarized. This paper also discusses the application of conventional breeding, molecular-marker-assisted breeding, gene editing, and other technologies in rice blast resistance breeding. The problem of accurately finding avirulence genes for R genes in current disease-resistant breeding is discussed and explored, aiming to improve rice blast resistance, agronomic traits, and yield in a sustainable way.

The type V CRISPR/Cas12f system, with its broad PAM recognition range, small size, and ease of delivery, has significantly contributed to the gene editing toolbox. In this study, enOsCas12f1 activity was detected during transient expression in rice protoplasts. The results showed that enOsCas12f1 exhibited DNA cleavage activity when it recognized TTN PAMs. Subsequently, we examined the gene editing efficiency of enOsCas12f1 in stably transformed rice plants, and the results showed that enOsCas12f1 could identify the TTT and TTC PAM sequences of the OsPDS gene, resulting in gene mutations and an albino phenotype. The editing efficiencies of TTT and TTC PAMs were 6.21% and 44.21%, respectively. Furthermore, all mutations were base deletions, ranging in size from 7 to 29 base pairs. Then, we used enOsCas12f1 to edit the promoter and 5′ UTR of the OsDREB1C gene, demonstrating that enOsCas12f1 could stably produce base deletion, mutant rice plants. Additionally, we fused the transcriptional activation domain TV with the dead enOsCas12f1 to enhance the expression of the target gene OsIPA1. Our study demonstrates that enOsCas12f1 can be utilized for rice gene modification, thereby expanding the toolbox for rice gene editing.