Chinese scientists reveal the molecular mechanism by which CRISPR acquires new spacer sequences
October 19, 2015 Source: Bio Valley
Window._bd_share_config={ "common":{ "bdSnsKey":{ },"bdText":"","bdMini":"2","bdMiniList":false,"bdPic":"","bdStyle":" 0","bdSize":"16"},"share":{ }};with(document)0[(getElementsByTagName('head')[0]||body).appendChild(createElement('script')) .src='http://bdimg.share.baidu.com/static/api/js/share.js?v=89860593.js?cdnversion='+~(-new Date()/36e5)];On October 15th, Cell Magazine published a research report on the acquisition stage of exogenous fragments in the CRISPR-Cas system by the research group Wang Yanli of the Institute of Biophysics of the Chinese Academy of Sciences. Title is Structural and Mechanistic Basis of PAM-dependent Spacer Acquisition in CRISPR-Cas Systems. This paper reveals the crystal structure of a series of complexes such as Cas1-Cas2-PAM-DNA complex, which proves that the new spacer acquires the complementary sequence dependent on PAM and provides an important structural biological basis for the mechanism of action of this process. .
Prokaryotes such as bacteria and archaea are constantly infected by external viruses and horizontal nucleic acid transfer. Clustered and regularly spaced short palindromic repeats and its accessory proteins (Cas) constitute an important immune defense for prokaryotes. System - CRISPR / Cas system. The defense process mainly includes three stages: (1) acquisition of the spacer, through the action of the core proteins Cas1 and Cas2, the nucleic acid fragment derived from the foreign invasion (ie, Spacer) is acquired and inserted into the CRISPR site. (2) CrRNA expression, which is transcribed into mature crRNA and forms a complex with the related Cas protein. (3) CrRNA interference, the mature crRNA-directed RNA-Cas protein complex recognizes an exogenous nucleic acid that is complementary to the sequence and eventually degrades it. The molecular and functional mechanisms of crRNA expression and interference have been revealed very thoroughly, and the acquisition process of the entire spacer has yet to be further studied.
Legend: Crystal structure of the E. coli Cas1-Cas2 protein and spacer precursor DNA complex with a bifurcated structure. Cas1-Cas2 specifically recognizes and binds DNA containing the PAM complementary sequence, thereby determining the length of the spacer precursor and inserting it into the CRISPR-Cas site.
Through in-depth research, Wang Yanli's research group analyzed the crystal structure of the complex of Cas1-Cas2 and various types of DNA, and proved that the exogenous nucleic acid fragment obtained by Cas1-Cas2 exists in a bifurcated conformation; Cas1-Cas2 The double-stranded portion is fixed and accurately quantified by two tyrosines, and the complementary sequence of the 3' single-stranded PAM (5'-CTT-3') is recognized in a sequence-specific manner, and is activated by Cas1, respectively At the 3' overhang at both ends, a length of 5 nt was cut, resulting in a 33 nt length DNA fragment; in the process, the combination with the exogenous nucleic acid fragment caused Cas1-Cas2 to undergo a "wing up" when it resembled a butterfly flying to The conformational change of "wing level" eventually inserts the acquired exogenous nucleic acid fragment into its own CRISPR site by a similar cut-copy method. The study found that Cas1-Cas2 recognizes the molecular mechanism of exogenous invading DNA, reveals how the length of the exogenous nucleic acid fragment is determined, and explains the respective functions of the core proteins Cas1 and Cas2 in this stage. Therefore, the results laid an important theoretical foundation for revealing the new mechanism of prokaryotic activity against the invasion of viruses and genetic material.
Wang Jiuli and Li Jiazhi (Ph.D.) of Wang Yanli's research group are the co-first authors of the article. The research was funded by the Ministry of Science and Technology, the National Natural Science Foundation, and the Chinese Academy of Sciences' Strategic Pilot Science and Technology Project (Class B). Research provides important technical support. (Bio Valley Bioon.com)
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