Structural dynamics of Cpf1 by FRET

Resumé

The RNA-guided endonuclease of class V Cpf1 (Clustered Regularly Interspaced Short Palindromic Repeats from
Prevotella and Francisella) is a central element in prokaryotic immune mechanism, which use a CRISPR-RNA (crRNA) to
locate and cleave viral DNA. Cpf1 allow genome-editing at a specific position specified by synthetic crRNA, hence a
promising therapeutic agent to treat gene deficiencies. Our current understanding of Cpf1 structure and function primarily
10 relies on crystal structures and cryoEM data, providing unique and invariant structures. In contrast to recent methods, the
scope of this project is to expand our understanding of the dynamic structure of Cpf1, and understand how conformational
changes and catalysis are related. To do this I used FRET (Förster Resonance Energy Transfer). Here, the work on this
projects is presented, with the aim of 1) understanding the conformational changes of Cpf1 in free-form, binary complex
(crRNA-bound) and tertiary complex (DNA-bound) in bulk, and correlating the conformational changes to intramolecular
15 distances, thus providing insight to the mechanism of DNA-cleavage and 2) calibrate the distance using dsDNA as a rigid
scaffold, preparing for single molecule FRET measurements on Cpf1. Ensemble measurements revealed conformational
changes of Cpf1 upon binding DNA, however the assay needs to be optimized further to extract distinct distances.
Spectrometric experiments revealed that excess dye in solution was a general problem in ensemble measurements,
interfering with the results. To validate and setup the calibration I did both ensemble and smFRET measurements on dual20
labelled dsDNA to address and eliminate the role of excess dye in solution. The inter-dye distance of dual-labelled dsDNA
was determined to be 62.72 ± 0.93 Å. The simulated distance with Monte Carlo simulations was found to be 61.6 Å. This
illustrates smFRET as a method to probe enzymatic motion, and thus could provide novel information about the mechanism
of DNA-cleavage in Cpf1, paving the way for future genome-editing.