The gene-editing CRISPR-Cas9 study yielded new results

November 9th, the research results of CRISPR-Cas9, a gene editing system, published online in
, an internationally renowned academic journal, in collaboration with the Huang Qiang Task Force of Fudan University's School of Life Sciences and the National Key Laboratory of Genetic Engineering, in collaboration with the Lu Daru Research Group. The results analyze the ACTIVE structure of DNA shearing in CRISPR-Cas9 by using the three-dimensional reconstruction method of single particles of cryoelectric mirrors, and important progress has been made in the study of DNA shear mechanism of CRISPR-Cas9.
Goingly, CRISPR-Cas9 technology, developed based on the bacterial accessible immune system, has become a revolutionary gene editing tool, and this "gene shear" can easily be used to efficiently and specifically edit genomic DNA, with great potential for application in biomedical fields. In order to understand the system's DNA shearing algorithm and guide the optimization of the system, over the past few years, research institutions have analyzed a number of "Cas9-sgRNA-DNA target chain" of the tri-composite crystal structure. However, the structure of these complexes does not fully reveal the true DNA shearing activity, and it is not clear how CRISPR-Cas9 cuts the molecular mechanism of DNA single strands through HNH and RUVC nuclease domains. Therefore, obtaining the cutting active structure of CRISPR-Cas9 is the key to reveal the DNA shearing process of the system.
in response to the above-mentioned research problems, Huang Qiang and Lu Daru research team as early as 2014 to consider the use of frozen electro-mirror methods to solve. They first constructed a ternate complex of SpCas9 enzymes, sgRNAs and DNA, and then analyzed the solution structure of the compound using a single-particle 3D reconstruction method of a frozen electroscope to obtain a frozen electromirror structure with a resolution of 5.2 E.The atomic model of the
complex structure shows that of all the structures analyzed, the HNH enzyme activity center of the complex is closest to the cutting point of the DNA chain, and molecular dynamics simulations and point mutation experiments show that the catalytic amino acids of the HNH and RUVC nuclease activity centers of the complex can form the desired composition of the cutting reaction with the DNA deconstruction single strand. Therefore, the compound structure obtained by the study is the DNA shear activation structure of CRISPR-Cas9, which provides the key active structure information for the comprehensive disclosure of the shear mechanism, and provides an important structural biochemical basis for the application of protein engineering technology to optimize the system and reduce its off-target effect. At present, under the guidance of the resulting 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.
is understood that the doctoral student Wai Cong and the master's student Li Jian are the first authors of the thesis, and Professor Huang Qiang and Professor Lu Daru are the co-authors of the newsletter. The research team used the electroscope platform of the National Protein Science Center (Shanghai) to collect the image of the frozen electroscope, and completed the three-dimensional reconstruction and atomic model construction using the electro-mirror image analysis and molecular modeling platform established by the team. The research projects have been supported by projects such as the National Natural Science Foundation of China. (Source: Science Network Huang Xin)