Programme

Structural biology enabled substantial advances on understanding how gene editing machineries operate across the three domains of life. In this talk, I will present cryo-electron microscopy (cryo-EM) studies of key gene editing machines, highlighting how conformational heterogeneity and intrinsic molecular flexibility underpin their biological function and engineering potential.

Early cryo-EM analyses revealed that NHEJ factors populate ensembles of dynamic states that enable recognition and processing of DNA ends^1-3. Later work on CRISPR effector complexes also highlighted pronounced structural plasticity, with macromolecular complexes captured as coexisting conformations rather than single static structures^4-6. For ΦC31 integrase, high-resolution cryo-EM structures illuminate large-scale domain rearrangements that drive unidirectional, site-specific recombination, providing a structural framework for understanding specificity and irreversibility⁷.

These insights have been enabled by recent advances in cryo-EM hardware as well as by developments in image processing and heterogeneity analysis software that allow continuous flexibility and transient intermediates to be resolved. Together, these studies demonstrate how embracing conformational variability, rather than averaging it out, is essential for mechanistic understanding of gene editing systems and for the rational development of next-generation genome engineering tools across all domains of life.

1. Morris, E.P., Rivera-Calzada, A., da Fonseca, P.C., Llorca, O., Pearl, L.H., and Spagnolo, L. (2011). Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic Acids Res 39, 5757-5767. 10.1093/nar/gkr146
2. Rivera-Calzada, A., Spagnolo, L., Pearl, L.H., and Llorca, O. (2007). Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs. EMBO Rep 8, 56-62. 10.1038/sj.embor.7400847
3. Spagnolo, L., Rivera-Calzada, A., Pearl, L.H., and Llorca, O. (2006). Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol Cell 22, 511-519. 10.1016/j.molcel.2006.04.013
4. Cannone, G., Kompaniiets, D., Graham, S., White, M.F., and Spagnolo, L. (2023). Structure of the Saccharolobus solfataricus type III-D CRISPR effector. Curr Res Struct Biol 5, 100098. 10.1016/j.crstbi.2023.100098
5. Rouillon, C., Zhou, M., Zhang, J., Politis, A., Beilsten-Edmands, V., Cannone, G., Graham, S., Robinson, C.V., Spagnolo, L., and White, M.F. (2013). Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol Cell 52, 124-134. 10.1016/j.molcel.2013.08.020
6. Zhang, J., Rouillon, C., Kerou, M., Reeks, J., Brugger, K., Graham, S., Reimann, J., Cannone, G., Liu, H., Albers, S.V., et al. (2012). Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell 45, 303-313. 10.1016/j.molcel.2011.12.013
7. Sun, Y.E. et al. bioRxiv 10.1101/2025.05.02.651858v2