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Updated: Sep 11, 2025
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Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
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Structural insights into Type II-D Cas9 and its robust cleavage activity
Kangkang Wang1,2,3, Jiuyu Wang1,2, Xiaoqi Yang1,2,3
1State Key Laboratory of RNA Innovation, Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Nature Communications
|August 11, 2025
Summary
A novel compact Cas9 variant, NsCas9d, shows strong DNA cleavage and produces staggered ends. This Cas9d enzyme recognizes a specific PAM and offers potential as an advanced genome-editing tool.
Area of Science:
- Molecular Biology
- Genomics
- Structural Biology
Background:
- Type II-D Cas9 proteins (Cas9d) are smaller than other Cas9 types.
- Understanding Cas9 diversity is key for genome editing tool development.
Purpose of the Study:
- To characterize NsCas9d from Nitrospirae bacterium RBG_13_39_12.
- To investigate its DNA cleavage activity, structure, and PAM recognition.
Main Methods:
- In vitro dsDNA cleavage assays.
- High-resolution cryo-electron microscopy (cryo-EM) of NsCas9d-sgRNA-dsDNA complex.
- Protospacer adjacent motif (PAM) analysis.
Main Results:
- NsCas9d exhibits robust in vitro dsDNA cleavage activity, comparable to SpCas9.
- NsCas9d generates 3-nucleotide staggered overhangs, unlike blunt ends from typical Cas9.
- NsCas9d recognizes the 5'-NRG-3' PAM, with 5'-NGG-3' being most efficient.
- Cryo-EM structure reveals DNA strand positioning in catalytic pockets.
Conclusions:
- NsCas9d is a compact and efficient genome-editing tool with unique cleavage properties.
- Its structure and sgRNA suggest Cas9d may represent evolutionary intermediates.
- NsCas9d offers a promising alternative for genome editing applications.
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