Visualization Of The CRISPR-Cas9 Genetic Engineering Mechanism


A technique used in genetic engineering is the CRISPR-Cas9 nuclease system. DNA can be cut at a desired site using this system, where genes can be deleted or added. The site is targeted by a 'guide RNA' molecule bound to the Cas9 protein. A team led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo visualized the dynamics of the CRISPR-Cas9 complex, using high-speed atomic force microscopy.

CRISPR is the acronym for "clustered regularly interspaced short palindromic repeats," and refers to a set of bacterial DNA sequences containing fragments of the DNA of viruses that have earlier infected the bacteria. "Cas" are CRISPR-associated genes. Among these "Cas9" has two nuclease domains (Nucleases are enzymes capable of cleaving DNA and RNA).

A tool for genetic engineering has been developed using the CRISPR-Cas9 complex to act as 'molecular scissors'. Cas9 binds to a guide RNA molecule that can target the selected site on DNA. Using high-speed atomic force microscopy, Shibata and colleagues studied the dynamics of the CRISPR-Cas9 complex to gain insights into its mechanism.

Atomic force microscopy (AFM) is an imaging technique in which the image is captured through a horizontal scanning motion controlled by piezoelectric elements and vertical motion which provides a height profile of the sample's surface.

A high-speed form of AFM (HS-AFM) can be used to produce movies of a sample's activity in real time, which has been used to study protein dynamics. The researchers used this set-up which can provide extremely fast, repeated scans which were then converted into movies – of the CRISPR-Cas9 biomolecules taking part in the molecular scissoring action.

When Cas9 was studied without and with RNA attached (Cas9-RNA), the scientists found that the former could adopt various conformations. The latter (Cas9-RNA) however, had a conformationally-stable fixed, two-lobe structure, signifying the ability of the guide RNA.

Shibata and team looked at how the stabilized Cas9-RNA complex targets DNA and found that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA. They found that the DNA target ('DNA interrogation') is attained through 3D diffusion of the Cas9-RNA complex.

Lastly, the researchers visualized the dynamics of the cleavage process itself and identified how the region of 'molecular scissors' of CRISPR-Cas9 undergoes conformational fluctuations after Cas9-RNA locally unwinds the double-stranded DNA.

The work of Shibata furthers our understanding of the CRISPR-Cas9 genome-editing mechanism. The researchers said: ".. this study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems."