The CRISPR-Cas system has become one of the most widely used tools in gene editing, allowing precise targeting and cleavage of DNA through RNA-guided Cas proteins. Recent advancements in bioinformatics and biochemistry have significantly expanded our understanding of the diversity within CRISPR systems, particularly in type V, which rely on the highly conserved RuvC nuclease domain for target cleavage. Cas12e (also known as CasX), a unique subtype within this family, has attracted significant research attention due to its small molecular size and potential for efficient gene editing. Two Cas12e orthologs, DpbCas12e and PlmCas12e, have been previously characterized with high conservation in both sequence and structure. A few months ago, the research groups of Chunlai Chen and Jun-Jie Gogo Liu collaborated to uncover the dynamic regulatory mechanisms governing the target search and cleavage processes of these proteins. However, a systematic study of the evolutionary diversity and functional differences within the Cas12e family has been lacking until now.
On December 30, 2024, groups of Jun-Jie Gogo Liu and Chunlai Chen from the School of Life Sciences at Tsinghua University, in collaboration with Dingding Su’s group at the Peking University Institute of Advanced Agricultural Sciences, published a groundbreaking study in Nature Communications titled “Cas12e Orthologs Evolve Variable Structural Features to Facilitate dsDNA Cleavage.” This study systematically identifies six novel Cas12e orthologs and provides in-depth structural and biochemical analysis to elucidate their distinct mechanisms in DNA target recognition, unwinding, and cleavage.
Using bioinformatics, the team identified six new Cas12e orthologs, ranging from 818 to 920 amino acids in size. Structural predictions revealed significant variations in the non-target-strand binding (NTSB) domains across these proteins (Figure 1). Biochemical assays confirmed that Cas12e proteins preferentially recognize T- or C-rich PAM sequences, demonstrating diverse DNA interference abilities. The researchers found that the cleavage efficiency of Cas12e orthologs is closely related to salt concentrations. Using single-molecule FRET experiments, the study further demonstrated that Cas12e orthologs exhibited higher R-loop formation and cleavage activity under low salt conditions, while high salt concentrations significantly inhibited their activity. This result reveals the regulatory role of salt concentration on Cas12e function and provides insights for optimizing their efficacy in complex environments.
Figure 1 . Structural and biochemical activity of novel CRISPR-Cas12e systems
Furthermore, the research team employed single-particle cryo-electron microscopy (cryo-EM) to resolve and analyze the structures of VemCas12e and LesCas12e ternary complexes. The analysis revealed that these orthologs’ different R-loop formation abilities under high salt conditions are attributed to specific structural elements responsible for DNA unwinding (Figure 2). Some Cas12e orthologs, such as PlmCas12e and DpbCas12e, contain a unique NTSB domain that promotes DNA unwinding and enhances cleavage activity under high salt conditions. In contrast, Cas12e orthologs lacking the NTSB domain, like VemCas12e and LesCas12e, rely on positively charged loops (such as Loop 1 and Loop 2) to unwind DNA but with less pronounced effects on R-loop formation and lower cleavage efficiency. Consistent with this, the NTSB domain in Plm2Cas12e lacks key residues involved in interactions with the non-target and target strands (NTS and TS), resulting in lower dsDNA cleavage activity under high salt concentrations. On the other hand, the OpbCas12e protein features a helix-loop structure in its RuvC nuclease domain that promotes R-loop formation, thus maintaining relatively higher dsDNA cleavage activity under high salt conditions (Figure 1). These structural differences underscore the biochemical diversity within the Cas12e family.
Figure 2 . Structural insights into Cas12e orthologs unwinding the dsDNA proximal to the PAM duplex
The study also presents an evolutionary perspective, showing how Cas12e orthologs have adapted to different environmental conditions by acquiring various structural features and diversifying their guide RNAs (Figure 3). These findings not only provide insights into the evolutionary mechanisms of the CRISPR-Cas system but also offer new strategies for optimizing Cas12e-based gene editing tools.
Figure 3 . Evolutionary diversity of Cas12e family proteins and gRNAs
The study highlights the potential of Cas12e orthologs, particularly PlmCas12e, which demonstrates the highest trans-cleavage activity, in nucleic acid detection. Reducing salt concentrations further enhances the trans-cleavage activity, underscoring the great potential of Cas12e proteins for molecular diagnostics and providing a theoretical foundation for optimizing their detection performance.
The study’s corresponding authors include Professor Jun-Jie Gogo Liu and Professor Chunlai Chen from Tsinghua University and Professor Dingding Su from Peking University. Tsinghua University’s PhD student Danyuan Li (2018 cohort), postdoc Shouyue Zhang (currently a researcher at the Institute of Microbiology, Chinese Academy of Sciences), PhD student Shuo Lin (2023 cohort), and PhD student Wenjing Xing (2018 cohort) are co-first authors. PhD student Yun Yang (2019 cohort) from Tsinghua University and Master’s student Fengxia Zhu (2022 cohort) from Peking University also contributed to the study. The research was funded by the Science and Technology Innovation - Major Project Youth Scientist Project, the National Key Research and Development Program, the Ministry of Agriculture and Rural Affairs of China, and the National Natural Science Foundation of China, among others.
Link to the articles:
https://doi.org/10.1038/s41467-024-54491-9
