Mobile genetic elements (MGEs) such as bacteriophages have evolved anti-CRISPR proteins (Acrs) to inhibit the adaptive immune system mediated by bacterial CRISPR-Cas. There have been recent reports that some non-phage-derived Acrs, such as AcrIIA17 and AcrIIA18, can inhibit Cas9 protein activity by modulating sgRNA [1], but the mechanism of their inhibition is unclear.
On December 10, 2021, the research group of Professor Zhang Heng of Tianjin Medical University published an article entitled"Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18 in the journal OfNucleic Acids Research, which explained in detail the mechanisms of AcrIIA17 and AcrIIA18 inhibiting CRISPR-Cas systems.
The CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CAS) and is an adaptive immune system of bacteria and archaea that resists the invasion of foreign mobile genetic elements. CRISPR-Cas systems can be divided into six types [2], among which gene editing technology based on type II CRISPR-Cas9 systems has been widely used in basic research and translational applications. So far, more than two dozen Acrs proteins targeting Cas9 systems have been discovered, and they inhibit Cas9 systems in different ways at different stages. Some of these inhibit CRISPR-Cas9 systems primarily by binding to Cas9 and hindering it from undergoing the necessary conformational changes, e.g., AcrIIA14, AcrIIC1, and AcrIIC3 bind to cas9's HNH nuclease domain, leaving it in a conformation without catalytic activity. There are also Acrs that prevent substrate DNA binding by mimicking the PAM sequence, binding to Cas9. Unlike the previously discovered type II Acrs, AcrIIA17-18 is mostly derived from non-bacteriophages and inhibits the CRISPR-Cas system by regulating sgRNA [1].
To investigate the regulatory mechanism of AcrIIA17, the authors used AlphaFold2 to predict the structure of AcrIIA17 and validated the interaction between AcrIIA17 and Cas9 by in vitro biochemical experiments such as DNA cleavage and EMSA, and found that AcrIIA17 and NmeCas9 acted stronger than SpyCas9. Using pull down and ITC experiments, the authors found that AcrIIA17 inhibited the assembly of the Cas9-sgRNA ribonucleoprotein (RNP) complex by binding to the bridge-like spiral (BH) domain of NmCas9, ultimately inhibiting the CRISPR-Cas9 system (Figure 1).
In order to study the inhibition mechanism of AcrIIA18, the authors first analyzed the high-resolution crystal structure of AcrIIA18 by X-ray crystallography. AcrIIA18 contains an N-terminal domain (NTD) and a C-terminal domain CTD. Since charged residues exposed to solvents may be involved in substrate binding and catalysis, the authors selected 13 charged residues on the surface of the NTD and CTD domains for point mutation and functional assessment, and found that V-shaped pockets on NTDs were critical to AcrIIA18's inhibitory effect. Through a series of biochemical experiments such as in vitro RNA cleavage, the authors found that AcrIIA18 can cleave the spacer region of sgRNA to 15nt, and the cleaved sgRNA is not enough to activate the endonuclease activity of Cas9, thereby inhibiting the CRISPR-Cas9 system (Figure 2).
Figure 2
In summary, the study elucidates the molecular mechanisms of AcrIIA17 and ACRIA18 inhibiting crispr-Cas9 systems from the perspective of biochemistry and structural biology. AcrIIA17 binds to the Cas9 bridge-like spiral (BH) domain to inhibit the assembly of the Cas9-sgRNA ribonucleoprotein (RNP) complex; AcrIIA18 inhibits the CRISPR-Cas system by cleaving the sgRNA. The results not only provide new insights into the mechanism by which Type II Acrs inhibits the CRISPR-Cas9 system, but may also provide a theoretical basis for the development of CRISPR-Cas9 system regulation tools.
Professor Zhang Heng and Dr. Bank of the School of Basic Medical Sciences of Tianjin Medical University are the co-corresponding authors of this article, and Wang Xiaoshen and Li Xuzichao, PhD students of the Class of 2021, are the co-first authors of this article.
Original link:
https://doi.org/10.1093/nar/gkab1197
Model Maker: Eleven
bibliography
1. Mahendra, C., Christie, K.A., Osuna, B.A., Pinilla-Redondo, R., Kleinstiver, B.P. and Bondy-Denomy, J. (2020) Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nature microbiology, 5, 620-629.
2. Mohanraju, P., Makarova, K.S., Zetsche, B., Zhang, F., Koonin, E.V. and Van der Oost, J. (2016) Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science, 353.
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