Analytical Tools Reveal Molecular Mechanism of Gene-Editing Tool

Despite the widespread usage of CRISPR/Cas9, DNA cleavage at off-target sites that resemble the target sequence continues to be a pervasive problem that remains poorly understood mechanistically. To solve the problem of off-target DNA cleavage, investigators at the University of Copenhagen (Denmark) utilized cryo-electron microscopy (cryo-EM) and single-molecule FRET (fluorescence resonance energy transfer) to dissect the reaction steps of DNA targeting by Cas12a (also known as Cpf1 or centromere and promoter factor 1).

Cryo-EM is an analytical technique that provides near-atomic structural resolution without requirements for crystallization or limits on molecular size and complexity imposed by the other techniques. Cryo-EM allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment while integrating multiple images to form a three-dimensional model of the sample.

Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. FRET can be used to measure distances between domains in a single protein and therefore to provide information about protein conformation.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

CRISPR/Cpf1 differs from CRISPR/Cas9 in a number of key ways. Cpf1 is much smaller than the Cas9 enzyme, which makes it easier to package inside a virus and therefore easier to deliver to cells. It also recognizes a different sequence of DNA than Cas9 does, which provides greater flexibility in terms of use.

In a paper published in the November 29, 2018, online edition of the journal Cell the investigators revealed cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sensed the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provided the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrated why Cas12a cut its target DNA and unleashed unspecific cleavage activity, degrading ssDNA (single strand DNA) molecules after activation.

"If we compare CRISPR to a car engine, what we have done is to make a complete three-dimensional map of the engine and thus gain an understanding of how it works. This knowledge will enable us to fine-tune the CRISPR engine and make it work in various ways – as a Formula One racer as well as an off-road truck", said senior author Dr. Guillermo Montoya, research director and group leader at the protein structure and function program at the University of Copenhagen.

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