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Harbin Institute of Technology

Huang Zhiwei research group published a paper in Nature that revealed the molecular mechanism of CRISPR-SpyCas9 inhibition by An anti-CRISPR protein

Updated: 2017-5-6

Reported by: Wang Ji
Translated by: Yu Tianyu
Edited by: Garrick Jones

Recently, a research group leaded by Professor Huang Zhiwei from the school of life, revealed the molecular mechanism CRISPR-SpyCas9 inhibition by An anti-CRISPR protein through a study,  which has provided a structural basis for the time of design, space-specific, conditionally precise control active tool of SpyCas9 gene editing. On April 27th, the research result has published online Nature as a paper which entitled “Structural basis of CRISPR-SpyCas9 inhibition by An anti-CRISPR protein”.

The CRISPR-Cas system is a bacterial-encoded adaptive immune system used to protect bacteria from phage infection, which cleaves viral DNA or RNA from crRNA-directed Cas nuclease to protect against viral infection. Due to the ability of the CRISPR-Cas system to combine with sgRNA to efficiently and easily "edit" the purpose of DNA, the Streptococcus Cas9 (SpyCas9) system, one of the CRISPR II-A subtypes, has been widely used in biomedical research worldwide as well as gene therapy, to do DNA knockout, activation, modification, mutation, etc. of the the cell, tissue or individual. The system has become the most important and most widely used gene editing tool.

However, how to reduce the genetic editing off-target effect which SpyCas9 over-activation or long-term activity brings, and how to precisely control the  time, space or conditions of SpyCas9 activity are important and challenging scientific issues that in current SpyCas9 system for cell or tissue gene editing, gene therapy and need to be addressed urgently. Previous studies have found that a class of anti-CRISPR gene AcrIIA4 from Listeria monocytogenes can inhibit the gene editing activity of SpyCas9 in cells. However, the molecular mechanism of these Anti-CRISPR genes in inhibiting SpyCas9 activity is unclear.

In order to investigate whether AcrIIA2 or AcrIIA4 can combine SpyCas9 directly, the research group first established an in vitro biochemical study system. It was found that Anti-CRISPR protein AcrIIA2 or AcrIIA4 combines with the SpyCas9-sgRNA complex directly. Interestingly, AcrIIA2 or AcrIIA4 only interacted with SpyCas9 which contains sgRNA and did not interact with SpyCas9 alone; further experiments showed that AcrIIA2 or AcrIIA4 was able to directly inhibit SpyCas9-mediated cleavage of the target DNA.

In order to study the molecular mechanism of AcrIIA4 direct inhibition of SpyCas9 activity, the SpyCas9-sgRNA-AcrIIA4 complex was purified by the research group, and the crystal structure of SpyCas9-sgRNA-AcrIIA4 complex was analyzed through structural biological way. The structure revealed that the AcrIIA4 protein conformation is a new fold that combines the three regions of SpyCas9 CTD, TOPO and RuvC to form a groove area. The groove region is the binding site of the substrate PAM DNA on SpyCas9. In addition, the contact of the Loop with the RuvC site from the β1 folded sheet of AcrIIA4 also enhanced the inhibitory effect of AcrIIA4 on SpyCas9. The above observations show that AcrIIA4 and the substrate PAM DNA compete with SpyCas9 and AcrIIA4 have stronger affinity for the substrate PAM DNA and further support the results of structural observation. The next biochemical test results further confirmed that AcrIIA4 binds to SpyCas9 to inhibit the substrate PAM DNA binding to SpyCas9, thereby antagonizing the activity of SpyCas9.

Significantly, the positions of the acidic amino acids Asp14, Asp37, Glu40, Asp69 and Glu70 of AcrIIA4 were completely consistent with the phosphate backbone positions of the PAM DNA bound to SpyCas9, and the amino acids of AcrIIA4 were directly linked to PAM on the SpyCas9 PI domain DNA recognition amino acid Glu1108, Ser1109, Ser1216, Lys1200, Arg1335 and Arg1333. These structural observations reveal that AcrIIA4 binds to SpyCas9 by simulating PAM DNA. The AcrIIA4 binding amino acids of the SpyCas9 protein are conserved in the same subtype N. meningitidis Cas9 (NmeCas9) and are not conserved in the different subtypes II-C Listeria monocytogenes Cas9 (LmoCas9), well thus explaining why AcrIIA4 can inhibit LmoCas9 Of the activity, but can not inhibit the activity of NmeCas9.

Through further structural comparison, analysis, subject group found that the formation of SpyCas9-sgRNA complex was not found on the SpyCas9 alone, and the SpyCas9 conformation was significantly changed, and the AcrIIA4 binding site was assembled to explain the formation of the SpyCas9-sgRNA complex. The initial biochemical study of this project shows that AcrIIA4 binds only to SpyCas9-sgRNA complexes without binding to SpyCas9 alone. The results are also consistent with the presence of SpyCas9 alone in the bacterial cells, whereas the SpyCas9-sgRNA complex is consistent with the predominant form. The SpyCas9-sgRNA complex is also formed when the phage is "discovered" by the bacterial CRISPR immune system and will be "disturbed", so this stage (SpyCas9-sgRNA complex phase) is a phage-inhibiting bacterial adaptive immune system (CRISPR -Cas9) being the most appropriate period.

The molecular mechanism of the anti-CRISPR protein AcrIIA4 which inhibits the activity of SpyCas9 has not only important scientific significance to reveal the co-evolutionary molecular mechanism of the bacteriological immune system (CRISPR-Cas9) and the "anti-CRISPR" , but also provides a structural basis for design time, space-specific, or conditionally precise control of the editing activity of SpyCas9 gene. The results were followed by the results of the study of pathogen-host interaction and gene editing in CRISPR-Cpf1 (Nature, 2016; RNA Biology, 2017) and CRISPR-C2c1 (Cell Research, 2017) An important research result.

Professor Huang Zhiwei is the corresponding author of this research paper, and doctoral students Dong De, Guo Minghui and master graduate student Wang Sihan in the team is the tied first author  of this paper. Post-doctoral Zhu Yuwei, master Wang Shuo, Xiong Zhi, Yang Jianzeng, undergraduate Xu Zengliang involved in part of the research work. All research work done at HIT. Shanghai Synchrotron Radiation Center provides crystal data collection supporting. The project is funded by the National Natural Science Foundation of China, Harbin University of young scientists studio and other funds.



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