3 years ago

Structure Of The Cpf1 Endonuclease R-Loop Complex After Target DNA Cleavage

P., G., Stella, S., Alcon, Montoya
Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool. To provide insight into its DNA targeting mechanism, we 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 45{degrees} 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. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 A away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a rail shape and flap-on conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a septum that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNA nuclease sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system.

Publisher URL: http://biorxiv.org/cgi/content/short/122648v1

DOI: 10.1101/122648

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.