Insights into the compact CRISPR–Cas9d system

Jie Yang, Tongyao Wang, Ying Huang, Zhaoyi Long, Xuzichao Li, Shuqin Zhang, Lingling Zhang, Zhikun Liu, Qian Zhang, Huabing Sun, Minjie Zhang, Hang Yin, Zhongmin Liu () and Heng Zhang ()
Additional contact information
Jie Yang: Tianjin Medical University
Tongyao Wang: Tianjin Medical University
Ying Huang: Southern University of Science and Technology
Zhaoyi Long: Tianjin Medical University
Xuzichao Li: Tianjin Medical University
Shuqin Zhang: Tianjin Medical University
Lingling Zhang: Tianjin Medical University
Zhikun Liu: Tianjin Medical University
Qian Zhang: Tianjin Medical University
Huabing Sun: Tianjin Medical University
Minjie Zhang: Tianjin Medical University
Hang Yin: Tianjin Medical University
Zhongmin Liu: Southern University of Science and Technology
Heng Zhang: Tianjin Medical University

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: Abstract Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo-EM structures of the Cas9d–sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated the PAM recognition and DNA cleavage mechanisms of Cas9d. Structural comparisons revealed that at least 17 base pairs in the guide–target heteroduplex is required for nuclease activity. Beyond its typical role as an adaptor between Cas9 enzymes and targets, the sgRNA also provides structural support and functional regulation for Cas9d. A segment of the sgRNA scaffold interacts with the REC domain to form a functional target recognition module. Upon target binding, this module undergoes a coordinated conformational rearrangement, enabling heteroduplex propagation and facilitating nuclease activity. This hybrid functional module precisely monitors heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpyCas9. Moreover, structure-guided engineering in both the sgRNA and Cas9d protein led to a more compact Cas9 system with well-maintained nuclease activity. Altogether, our findings provide insights into the target recognition and cleavage mechanisms of Cas9d and shed light on the development of high-fidelity mini-CRISPR tools.

Date: 2025
References: Add references at CitEc
Citations:

Downloads: (external link)
https://www.nature.com/articles/s41467-025-57455-9 Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57455-9

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-025-57455-9

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().