Type III CRISPR-Cas system provides prokaryotes with adaptive defence against viruses. It was recently noted that these systems employ specialized cell signalling pathways to combat infections. When CRISPR-Cas effector complex recognizes foreign RNA it starts to synthesize cyclic oligoadenylates from ATP [1]. These molecules activate diverse CRISPR ancillary proteins which consist of signal recognition domain and effector domain: RNase, DNase, ATPase, protease or others [2]. Functions and structures of several effector domains have been elucidated: for example, the RNase activity of HEPN domains [3, 4] and the DNase activity of restriction endonuclease-like effectors [5, 6]. However, many other effector domains have not yet been studied. Recent sequence analysis revealed that a group of such effectors are similar to translation-inhibiting toxins from bacterial toxin-antitoxin systems [2]. In this study we present crystallization results for toxin related to CRISPR-Cas system and discuss possible future strategies.
CRYSTALISATION OF CRISPR-CAS RELATED TOXIN
Konstanty Keda1, Giedre Tamulaitiene1, Irmantas Mogila1, Virginijus Siksnys1, Gintautas Tamulaitis1
1 Institute of Biotechnology, Life Science Center, Vilnius University, Lithuania
[1] Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. and Siksnys, V. (2017) A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science, 357(6351), р. 605-609.
[2] Makarova, K.S., Timinskas, A., Wolf, Y.I., Gussow, A.B., Siksnys, V., Venclovas, Č. and Koonin, E.V. (2020). Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic acids research, 48(16), p. 8828-8847.
[3] Smalakyte, D., Kazlauskiene, M., F. Havelund, J., Rukšėnaitė, A., Rimaite, A., Tamulaitiene, G., Færgeman, N.J., Tamulaitis, G. and Siksnys, V. (2020) Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic acids research, 48(16), p. 9204-9217.
[4] Garcia-Doval, C., Schwede, F., Berk, C., Rostøl, J.T., Niewoehner, O., Tejero, O., Hall, J., Marraffini, L.A. and Jinek, M. (2020) Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6. Nature communications, 11(1), p. 1-9.
[5] McMahon, S.A., Zhu, W., Graham, S., Rambo, R., White, M.F. and Gloster, T.M. (2020) Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate. Nature communications, 11(1), p. 1-11.
[6] Rostøl, J.T., Xie, W., Kuryavyi, V., Maguin, P., Kao, K., Froom, R., Patel, D.J. and Marraffini, L.A. (2021) The Card1 nuclease provides defence during type-III CRISPR immunity. Nature, p.1-9.