PHAges REvenge: structural and functional analyses of anti CRISPR-Cas9 proteins – PHARE
Phages Revenge : exploring the diversity of molecular mechanisms used by proteinaceous inhibitors of CRISPR-Cas9 bacterial immunity
Structural and functional analyses of anti-CRISPR-Cas9 proteins, identified in virulent bacteriophages infecting Streptococcus thermophilus, with different modes of inhibition
How diverse are the molecular mechanisms deployed by anti-CRISPR-Cas9 proteins?
Anti-CRISPR-Cas9 proteins are used by bacteriophages (phages) to defend themselves against the CRISPR-Cas9 adaptive immune system of their hosts. The striking sequence diversity of anti-CRISPR-Cas9 proteins identified so far and the absence of homologs with known function raise questions relating to their mode of action. In particular, we wonder whether the anti-CRISPR-Cas9 sequence diversity results in diverse anti-CRISPR-Cas9 structures and functions. Deciphering the molecular mechanisms of these proteins is essential to better understand phage-host interactions and the co-adaptation of their attack and defense mechanisms. Moreover, these proteins have rapidly garnered interest as they form a promising reservoir of biotechnological tools to fine-tune CRISPR-Cas9-based gene edition, and as useful addition to phage therapy.<br />In this context, our main objective is to elucidate the molecular mechanisms of the anti-CRISPR-Cas9 proteins AcrIIA5 and AcrIIA6, identified in virulent phages infecting Streptococcus thermophilus, with different inhibition profiles. While AcrIIA5 is a broad-spectrum anti-CRISPR protein that inactivates different CRISPR-Cas9 systems, including Streptococcus pyogenes CRISPR-Cas9 largely used in Cas9-based gene edition, AcrIIA6 specifically inhibits CRISPR1-Cas9 from S. thermophilus (St1Cas9). Moreover, inspired by these proteinaceous inhibitors of CRISPR-Cas9, we also aim at producing nanobodies (antigen-binding domains from camelid antibodies) inactivating CRISPR-Cas9 that could be used as regulators of Cas9-based gene edition tools.
Our proteins of interest are recombinantly produced in Escherichia coli, purified to homogeneity, and characterized using biochemical and biophysical technics (light scattering coupled to size exclusion chromatography, in vitro DNA cleavage assays, analyses of macromolecular interactions). Structural analyses are performed by X-ray crystallography and/or cryo-electron microscopy. Nanobodies are generated by the NAbGen Technology platform (https://nabgen.org/technologie/nanobodies/). The functional impact of anti-CRISPR proteins are assessed in cellular environments by our collaborators Sylvain Moineau and Yannick Doyon (Université Laval Québec).
We showed that the AcrIIA6 molecular mechanism is unique. AcrIIA6 acts as an allosteric inhibitor that binds to a St1Cas9 region that is well separated from its functional DNA-binding and nuclease active sites. By doing so, AcrIIA6 affects St1Cas9 conformational dynamics, which reduces St1Cas9 binding affinity for DNA and prevents St1Cas9 binding to its target within cells. Additionally, AcrIIA6 induces St1Cas9 dimerization, which makes it a potent inhibitor enabling rapid phage propagation. These findings led us to identify a natural variant of St1Cas9 resistant to AcrIIA6. This St1Cas9 variant harbors different amino acids in the AcrIIA6-binding site, thereby illustrating anti-CRISPR-driven mutational escape and molecular diversification of Cas9 proteins.
Our project of structural and functional analyses of anti-CRISPR-Cas9 proteins unveils the diversity of their molecular mechanisms to inactivate the bacterial CRISPR-Cas9 immunity. Our ongoing work on the anti-CRISPR protein AcrII5 indicates that this inhibitor has a nuclease enzymatic activity that affects the function of different CRISPR-Cas9 systems. It would be the first example of an enzymatic anti-CRISPR-Cas9 protein.
1. Fuchsbauer, O., Swuec, P., Zimberger, C., Amigues, B., Levesque, S., Agudelo, D., […], Goulet A. (2019). Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6. Mol. Cell 76, 922-937.e7.
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2. Agudelo D, Carter S, Velimirovic M, Duringer A, Levesque S, Rivest JF, Loehr J, Mouchiroud M, Cyr D, Waters PJ, Laplante M, Goulet A, and Doyon Y. (2020) Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9, Genome Res., 30(1):107-117
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3. Hardouin, P., Goulet, A. (2020). Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?, Biochemical Society Transactions, 48(2):507-516
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Bacterial viruses (bacteriophages or phages) are now recognized as the most abundant and diversified biological entities on Earth. Bacterial cells have survived attacks by phages by evolving sophisticated defense strategies to thrive in virus-rich ecosystems, including the well-known CRISPR-Cas9 system. In parallel, phages have developed several tactics to overcome these mechanisms, including anti-CRISPR proteins (Acr), leading to a so-called biological arms race. While the first Acrs were discovered in prophages (phages lying dormant in a CRISPR-carrying host) to mainly avoid host self-targeting, they were thought to be largely absent from virulent phages (phages that cannot become prophages). Very recently, our collaborator has identified two unique families of Acrs encoded in strictly virulent phages that evaded CRISPR-Cas9 immunity in Streptococcus thermophilus. The tremendous sequence diversity of the Acrs identified so far, their large spreading in phages, and the absence of homologs of known function add a fascinating part to the CRISPR-Cas story. These notable features raise intriguing questions about their molecular mechanisms and potential extra roles, beyond inhibiting CRISPR-Cas9 immunity, in phage biology. Moreover, Acrs are the first examples of off-switches for CRISPR-Cas9-based genome manipulation and hence offer great promise as tools to fine-tune gene edition. The anti-CRISPR field of research is still in its infancy; with PHARE (PHAge REvenge) we propose an innovative program of research focused on three main objectives. First, deciphering the modes of action at the molecular level of newly found Acrs in virulent streptococcal phages that inactivate CRISPR-Cas9 system. Second, examining the effects of acrs gene inactivation on phage biology. And third, generating nanobodies (single immunoglobulin domain derived from camelid antibodies) as ‘Acr-like’ CRISPR-Cas9 inhibitors and, if possible, tandems of non-overlapping nanobody and Acr as biotechnological tools for CRISPR-Cas9-based applications. This research will benefit fundamental knowledge in many life sciences fields (virology, microbiology, evolution, biochemistry, structural biology), and also help development of necessary off-switches for gene editing technologies in biomedical research and therapeutics. Thus overall, the project’s outcome may have long term positive societal and economic impacts, beyond the short and medium term scientific gains.
Project coordination
Adeline Goulet (Architecture et fonction des macromolécules biologiques)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
AFMB Architecture et fonction des macromolécules biologiques
Help of the ANR 265,005 euros
Beginning and duration of the scientific project:
December 2018
- 36 Months
