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Updated: Nov 6, 2025
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
34.7K
[Structure-based optimization and design of CRISPR protein xCas9].
Dongmei Xue1, Haixia Zhu1, Wenhao Du1
1School of Life Sciences, Fudan University, Shanghai 200438, China.
Summary
Researchers optimized the Streptococcus pyogenes Cas9 (SpCas9) genome editing tool, creating a new yCas9 mutant. This enhanced CRISPR/Cas9 system offers broader PAM recognition and reduced off-target effects for biomedical applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Streptococcus pyogenes Cas9 (SpCas9) is a key genome editing tool.
- SpCas9 has limitations including a narrow protospacer adjacent motif (PAM) range and off-target effects.
Purpose of the Study:
- To rationally optimize the xCas9 mutant derived from SpCas9 using directed evolution.
- To develop a new Cas9 variant with expanded PAM recognition and reduced off-target activity.
Main Methods:
- Applied energy minimization using Rosetta to optimize Cas9 3D structure.
- Designed combinatorial mutations based on xCas9 evolution and selected optimal mutants via free energy ranking.
- Verified yCas9 performance through DNA cleavage experiments and molecular dynamics simulations.
Main Results:
- Developed yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition (NG, GAA, GAT).
- yCas9 demonstrated low off-target DNA cleavage activity with mismatched sgRNA.
- Molecular dynamics simulations elucidated PAM recognition and off-target mechanisms for SpCas9, xCas9, and yCas9.
Conclusions:
- The yCas9 mutant provides a versatile gene editing tool with improved PAM specificity and reduced off-target effects.
- This optimized CRISPR/Cas9 variant has significant potential for biomedical applications.
- Findings offer theoretical guidance for future CRISPR/Cas9 protein engineering.
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