Conference How CRISPR-Cas endonucleases cut specific regions of the Genome

9 de July de 2017

The next 13th of July at 1.00p.m will be held the conference organized by the Department of Molecular Biology and the Faculty of Medicine of the University of Cantabria, given by Dr. Guillermo Montoya who will talk about the mechanisms of recognition and cutting of the DNA of the endonuclease of class 2 type V CRISPRCas system as a powerful genome editing tool.

Guillermo Montoya is Professor at the University of Copenhagen (UCPH) and Research Director of the Protein Structure & Function Programme at the Novo Nordisk Foundation Centre for Protein Research ( Montoya is also the Chairman of ISBUC, the Integrative Structural Biology Cluster at UCPH ( In the last years, his main research interests are the structural analysis of macromolecular complexes of the cell cycle such as the mammalian chaperonin CCT complex and the CMG complex, where he uses the combination of X-ray crystallography and electron microscopy to gain insight about their working mechanisms. He is also systematically pursuing the structure-function analysis of endonucleases, which are of great interest because of their potential applications in gene therapy. 

Dr. Montoya is member of international project evaluation panels and also reviews projects for European funding agencies (ERC, EU, SNF, DFG, NOW, MINECO, Welcome Trust) and serves as an associate editor and ad-hoc reviewer for high-profile journals. In addition, his group has been the receptor of Marie Curie, EMBO and HFSP postdoctoral fellows. He has been awarded the National Prize from the Fundación Mutua Madrileña (2009) and the V Health Sciences Prize from Caja Rural de GranadaMinisterio de Sanidad (2009) for his work in the redesign of genome editing enzymes. 

Summary of the Lecture: Structural Biology of Genome Editing: How CRISPR-Cas endonucleases cut specific regions of the Genome?

Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPRCas system, emerging as a powerful genome editing tool. To provide insight into its DNA targeting mechanism, they have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45º angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNA nuclease sites. Their study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system.

Five key papers:

Stella S, Alcón P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. (2017) May 31. doi: 10.1038/nature22398. 

Molina R, Stella S, Redondo P, Gomez H, Marcaida MJ, Orozco M, Prieto J, Montoya G. Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nature Structural & Molecular Biology (2015) Jan;22(1):65-72. doi: 10.1038/nsmb.2932. 

Mortuza G., Cavazza T., Garcia-Mayoral MF, Hermida D., Peset I., Pedrero J.G., Lyngsø, J. Marta Bruix, Pedersen, J.S, Vernos, I. and Montoya, G. The XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation. Nature Communications (2014) DOI: 10.1038/ncomms6072. 

Muñoz, I.G., Yébenes, H., Zhou M., Mesa, P. Serna, M. Bragado-Nilsson, Beloso, A., E. de Carcer G., Malumbres M., Robinson C.V., Valpuesta, J.M. & Montoya G. Crystal structure of the mammalian cytosolic chaperonin CCT in complex with tubulin. Nature Structural & Molecular Biology (2011) Jan; 18(1):14-9. 

P. Redondo, J. Prieto, I. Muñoz, A. Alibés, F. Strichter, L. Serrano, S., P. Duchateau, F. Paques, F. Blanco & G. Montoya. Molecular basis of recognition and cleavage of the human Xeroderma pigmentosum group C gene by engineered homing endonuclease heterodimers. Nature (2008). Nov 6;456(7218):107-1