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Molecular Biology

A breakthrough – CRISPR Cas9 imaged for the first time

CRISPR Cas9 from S.aureas, based on ID 5A XW protein database.
CREDIT: Thomas Splettstoesser

The discovery of CRISPR opened the pandora box of opportunities for scientists across the world to explore this gene editing tool further. But since scientists were not having any visuals of the CRISPR Cas9 enzyme, it was difficult to come up with any new discoveries or method of gene editing. Recently, researchers at the University of British Columbia and the University of Illinois have captured the 3-D image of the CRISPR Cas9 enzyme at DNA cleavage stage.

The scientist used a well-known technique named “Cryogenic Electron Microscopy”. The technique has been in news for long in the past few decades due to its ability to provide high-resolution images. When used it gave the detailed atomic-level visuals of the CRISPR Cas9 enzyme both prior and after cleavage of DNA strands. The research has been published on 8th July 2019 in the journal – “Nature Structural and Molecular Biology”

What is CRISPR?

CRISPR technology is inspired by the defense mechanism used in bacteria. Wherein any foreign invasion by infectious agents results in the accumulation of its genetic information within the bacterial genome. This information is passed on to the subsequent generation of the bacteria, and is located on the loci known as CRISPR, which stands for “Clustered Regularly Interspaced Short Palindromic Repeats”.  When the next time the bacteria are exposed to foreign invasion by a virus or other agent, its immune system recognizes the agent and get activated against it. This results in a cascade of signals which pump up the synthesis of guide RNA. This guide RNA interacts with an enzyme named as Cas9 (CRISPR associated protein 9) and forms an active complex (includes guide RNA, Cas9 and target DNA sequence). Cas9 is a restriction endonuclease enzyme and as soon as it binds to the target DNA it cleaves it at specific sites.

Why studying the image of CRISPR Cas9 is important?

The availability of CRISPR Cas9 image can bring revolution in the development of new gene-editing tools. Till now, one of the major obstruction in the scientific development of gene editing tool was the unavailability of the enzyme image. Thus, scientists were lacking a lot of information about the functioning of CRISPR Cas9 enzyme, especially after it cuts the DNA strands.

Scientists who are interested in modulating the activity of the CRISPR Cas9 enzyme or constructing mutant enzyme were not having sufficient information to start with. This obstructed the development of many new techniques and breakthrough discoveries. But since now we have a detailed image, the scientist can visualize the individual domains of the enzyme easily. This will allow them to understand the movement of the enzyme during the process of DNA strand cleavage.

How scientists made it possible?

Scientist knew that imaging CRISPR Cas9 is very important to understand how it actually works at a molecular level. For this, they compared many techniques and were very clear that imaging Cas9 require a different and high throughput imaging technique. Moreover, for the past few years, cryogenic electron microscopy (cryo-EM) has gained a lot of popularity due to its ability to give high-resolution images of biomolecules.

For imaging, the scientist used active Cas9 enzyme coupled with DNA and guide RNA. In order to activate the enzyme for cutting DNA strands, they added magnesium ions. The complex was then flash frozen for cryogenic electron microscopy and imaged to give atomic level CRISPR Cas9 visuals. Three different structural conformations were imaged when the enzyme was in a complex form with nucleic acids. It was observed that in two of the three images, the DNA strand was cleaved by the enzyme but Cas9 was still bound to it.

The entire process of CRISPR cleavage can be categorized into three stages:

1st Stage: In the initial step, the major domains of enzyme move over the DNA to assess if the sequence to be cleaved is proper. This stage was captured in the first snapshot of the cryo-EM.

2nd Stage: Under this stage, the Cas9 was imaged after the DNA strands have been cleaved. It was found that the enzyme hovers on the DNA sequence for proper cutting of the DNA sequence.

3rd Stage: At the final stage, when the DNA is cleaved successfully by the Cas9, it moves towards the end for departure.

Thus, the imaging using cryo-EM helped to scientist to picture the entire CRISPR Cas9 reaction.

The learning’s from the experiment

The high-resolution image of CRISPR Cas9 has marked the rise of new hope among scientific fraternity to uncover its hidden prospects.  The present study helped them to know many new properties of Cas9 interaction with the DNA and guide RNA. One of the most crucial observations was about the functioning of different domains and their interaction during the biochemical reaction. Some domains were swapping between ordered states to disordered states whereas others were stable. The domain switched between many conformational states which were not observed or assessed earlier. Thus, the visualization of Cas9 could be used to alter the targeting process in order to design new gene-editing tools. It has future applications for the treatment and preventions of various genetic diseases which involves DNA mutations such as cancer, cystic fibrosis and many more.



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