The DNA shear active structure of CRISPR-Cas9 was analyzed by using the three-dimensional reconstruction method of cryptoscope single particles.

Today, the research results of the gene editing system CRISPR-Cas9, conducted online by Huang Qiang Of fudan University's School of Life Sciences and the National Key Laboratory of Genetic Engineering, in collaboration with The Lu Daju Group, were published online in the internationally renowned academic journal Nature Communications.
the results of using the frozen electromirror single particle 3D reconstruction method to analyze the DNA shear active structure of CRISPR-Cas9, in CRISPR-Cas9 DNA shear mechanism research has made important progress.
now, CRISPR-Cas9 technology based on the bacterial acquired immune system has become a revolutionary gene editing tool, a "gene shear" that can easily efficiently and specifically cut and edit genomic DNA, with great potential for application in the biomedical field.
in order to understand the SYSTEM's DNA shearing mechanism and guide the optimization of the system, in the past few years, the research institutions have analyzed a number of "Cas9-sgRNA-DNA target chain" of the ternary complex crystal structure.
However, these complex structures do not fully reveal the true DNA shear active structure, and it is not clear how CRISPR-Cas9 cuts the molecular mechanism of DNA monostrands through hNH and RuvC nuclease domains.
, obtaining the shear active structure of CRISPR-Cas9 becomes the key to reveal the shear mechanism of DNA in the system.
in response to the above-mentioned research problems, Huang Qiang and Lu Daju research team as early as 2014 to consider the use of cryoscopy method to solve.
they first constructed a ternary compound of Cas9 enzyme (SpCas9), sgRNA and DNA, and then analyzed the solution structure of the complex with a three-dimensional reconstruction method of a single particle of the frozen electron mirror, and obtained a frozen electron structure with a resolution of 5.2 e. The atomic model of the
compound structure shows that in all the structures resolved, the HNH enzyme activity center of the complex is closest to the cutting site of the DNA chain, and the molecular dynamic simulation and point mutation experiments show that the catalytic amino acids at the HNH and RuvC nuclease activity centers of the complex can be conformations of the single-chain formation of DNA de-screwing.
, the composite structure obtained by the study is crispR-Cas9's DNA shear-activated conformation, which provides the key active structure information for the comprehensive disclosure of shear mechanism, and provides an important structural biochemical basis for optimizing the system with protein engineering technology and reducing its off-target effect.
currently, under the guidance of the acquired structure, the research team is using protein design methods to optimize the CRISPR-Cas9 system in order to develop a new gene editing system with low off-target effect and high editing efficiency.
it is understood that doctoral student Saphon and master's student Li Gan are the first authors of the thesis, and Professor Huang Qiang and Professor Lu Daju are side-by-side communication authors. The
research team used the electromirror platform of the National Protein Science Center (Shanghai) to collect frozen electron mirror images, and mainly used the electroscope image analysis and molecular modeling platform established by the research group to complete the three-dimensional reconstruction and atomic model construction.
related research projects have been supported by the National Natural Science Foundation of China and other projects.
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