CRISPR-Cas12a Gene Editing Technology and Its Application in Agricultural Production

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR- Cas) gene editing technology has not only revolutionized life sciences but also catalyzed transformative advancements in agriculture. As a critical branch of the CRISPR system, the CRISPR-Cas12a system exhibits unique molecular characteristics and distinct application potential in biological breeding and disease diagnosis compared to the classical CRISPR-Cas9 system. Unlike the Type II Cas9 system, the Type V Cas12a protein possesses a single RuvC-like nuclease domain, contrasting sharply with the dual HNH-RuvC nuclease domains of Cas9. Cas12a generates staggered double-strand breaks (DSBs) in target DNA while retaining the CRISPR RNA (crRNA) and Cas12a-formed "R-loop". The preservation of this R-loop constitutes the the structural basis for the collateral cleavage activity inherent to the CRISPR-Cas12a system, which underpins its utility in developing nucleic acid and small molecule detection technologies. Recognizing thymine-rich protospacer adjacent motifs (PAMs), CRISPR-Cas12a acts as a powerful complement to existing CRISPR-Cas systems. Its crRNA-dependent autonomous processing mechanism, distinct from the tracrRNA-dependent system of Cas9, offers superior advantages in multiplex gene editing. These features have driven breakthroughs in crop genetic improvement, including the successful development of disease-resistant and high-yield commercial crop varieties. In basic research, catalytically inactive Cas12a (dCas12a) fused with transcriptional regulators or epigenetic modifiers enables precise gene expression regulation without inducing DSBs. Furthermore, its integration with isothermal amplification techniques allows for visual disease detection. This review systematically introduced the CRISPR-Cas12a system from multiple perspectives: (1) classification of Type V Cas proteins, (2) mechanistic principles of Cas12a in bacterial immunity, and (3) functional domains of the Cas12a-crRNA complex. A comparative analysis between CRISPR-Cas12a and CRISPR-Cas9 was conducted across four dimensions: crRNA processing mechanisms, structural-functional features of Cas effectors, editing efficiency, and application scenarios. Additionally, the regulatory systems of CRISPR-dCas12a and CRISPR-dCas9 were evaluated regarding gene expression modulation, epigenetic editing, and base editing. The review also elucidated the molecular detection principles of CRISPR-Cas12a in targeting nucleic acids, proteins, and small molecules, as well as its agricultural applications in gene regulation, base editing, pathogen detection, disease diagnosis, and bio-breeding. With the emergence of safer non-DSB- dependent technologies such as prime editing, the CRISPR-Cas12a system was poised to play an increasingly vital role in crop precision breeding, livestock genetic improvement, and rapid clinical diagnostics. These advancemented promise innovative solutions to global food security challenges and infectious disease control, further cementing CRISPR-Cas12a as a cornerstone tool in agricultural biotechnology and molecular medicine.