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Updated: Jun 28, 2025
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Secondary Conformational Checkpoint in CRISPR-Cas9
Shuxin Zhao1,2, Jin Liu3, Zhicheng Zuo1,2
1College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China.
Abstract:
A specific checkpoint between target DNA binding and cleavage primarily governs the precision of Cas9 gene editing. Although various CRISPR-Cas9 variants have been developed to improve DNA cleavage accuracy, we still lack a comprehensive understanding of how they work at the molecular level. Herein, we have focused on studying the late-stage conformational transitions of Cas9 and an evolved Cas9 mutant (evoCas9) that start from the precleavage state. Our submilliseconds of dynamic simulations reveal that the presence of base mismatches leads the HNH nuclease domain of Cas9 to alter its principal functional modes of motion, thereby impairing its conformational activation. This observation suggests the existence of a secondary conformational checkpoint that fine-tunes the final DNA cleavage activation. Remarkably, evoCas9 is prone to deviating from the normal activation pathway with base mismatches. This is characterized by a noticeable shift in the positioning of the HNH domain and a significantly perturbed allosteric communication network within the enzyme. Therefore, the mutations evolved in evoCas9 also reinforce the secondary checkpoint in addition to the previously identified primary checkpoint, collectively ensuring this variant's high gene-editing accuracy. This mechanism should also apply to other Cas9-guide RNA variants with enhanced fidelity.
Insights
Researchers discovered a secondary checkpoint in Cas9 gene editing that enhances accuracy. Evolved Cas9 variants strengthen this checkpoint, improving precision by controlling DNA cleavage activation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- CRISPR-Cas9 gene editing precision is crucial but not fully understood at the molecular level.
- Existing CRISPR-Cas9 variants aim to improve DNA cleavage accuracy, yet their mechanisms require deeper investigation.
Purpose of the Study:
- To investigate the late-stage conformational dynamics of Cas9 and an evolved variant (evoCas9) in the precleavage state.
- To elucidate the molecular mechanisms underlying the enhanced accuracy of evoCas9.
Main Methods:
- Sub-millisecond dynamic simulations were employed to observe conformational transitions.
- Analysis focused on the HNH nuclease domain's motion and allosteric communication networks.
Main Results:
- Base mismatches disrupt Cas9's HNH nuclease domain motion, impairing conformational activation and suggesting a secondary checkpoint.
- evoCas9 exhibits altered activation pathways with mismatches, including HNH domain repositioning and perturbed allosteric networks.
- Mutations in evoCas9 reinforce both primary and secondary checkpoints, ensuring high gene-editing accuracy.
Conclusions:
- A secondary conformational checkpoint fine-tunes Cas9 DNA cleavage activation, particularly in the presence of base mismatches.
- The evolved Cas9 variant (evoCas9) utilizes enhanced checkpoint mechanisms for superior gene-editing fidelity.
- The identified mechanism likely applies to other high-fidelity Cas9-guide RNA variants.
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