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(Nanowerk News) For the first time, scientists were trying to improve the efficiency of CRISPR technology – a gene editing platform using an enzyme called Cas9 to precisely cut and edit specific sequences of DNA in a living cell – have captured the atomic level, three-dimensional images of the enzyme before and after DNA cleavage.
The images provide new structural information on how the enzyme works and will help researchers develop modified versions of the enzyme that can more effectively and accurately alter the targeted genes. Images and glimpses of Cas9 made from them are published in Nature Structural and Molecular Biology ("Cryo-EM structures reveal coordinated domain motions that govern the cleavage of DNA by Cas9").
CRISPR is a gene editing tool that allows scientists to remove unwanted genes or genetic material from DNA or add a desired sequence to a gene to alter its function or regulate its activity. CRISPR uses an enzyme called Cas9 that acts as scissors that cut a specific DNA sequence. Once the cuts are made on either side of the DNA, the cell initiates repair systems that join both ends of the DNA strands.
"One of the main obstacles to developing better gene editing tools using Cas9 is that we did not have any images of the enzyme after cutting DNA and did not have complete information. on the changes that this very important enzyme undergoes to execute the process. "Miljan Simonovic, badociate professor of biochemistry and molecular genetics at the University of Illinois at Chicago, and corresponding author of the journal.
Previous structural images of Cas9 have been obtained by X-ray crystallography, but this approach has limitations. To capture various states of the enzyme in crystalline form, the researchers use either the inactive version of Cas9 (a version of the enzyme that does not cleave the DNA), or the formation of crystals in them. conditions that do not allow cleavage of DNA. These processes can only produce images of the enzyme before it is cut.
"Researchers interested in modulating the activity of Cas9 or developing mutant enzymes that might work better did not have complete information to begin with, so that advancement did not occur. not as fast as desired, "said Simonovic. "But now we have a much clearer picture, and we even see how the main areas of the enzyme move during the reaction, which may be important for further exploration."
"It's exciting to see how Cas9 actually works to cut and edit DNA strands with such a high level of detail," said study co-author, Sriram Subramaniam, a professor of medicine at the University of California. 39, University of British Columbia. "These images provide us with valuable information to improve the efficiency of the gene modification process so that we can hope to more quickly and accurately correct mutations of DNA causing disease in the future. "
Simonovic knew that a different imaging technique was needed to really see Cas9 at work. Over the past decade, cryogenic electron microscopy, or cryo-EM, has become well known for its ability to image large, high-resolution molecules under conditions close to their natural environment.
Simonovic and his colleagues used active Cas9 as well as DNA and RNA guidance. Magnesium, which is needed to activate the enzyme for cleavage of DNA, was added and Cas9 complexes were frozen and imaged with the aid of cryo-EM.
The final results were snapshots of Cas9 trapped in three distinct structural arrangements while they were linked to nucleic acids. More remarkably, in two of the three states, the target DNA was cleaved, but the enzyme remained bound to it, allowing this latter stage of the biochemical reaction sequence to be observed with high resolution.
In the first state, the enzyme is captured before cutting DNA, its main areas "evaluating" if the sequence to cut is correct. In the second case, Cas9 was imaged almost immediately after cutting off the DNA. Practically, the active site of the enzyme floats above the cleavage in the strand of DNA. And, in the third state, the enzyme began its movement toward its departure from the cleaved DNA.
"We have discovered several new things about how Cas9 interacts with DNA and how it works." One of the most interesting is how many enzyme domains move in a concerted fashion and alternate between ordered and disordered states during the reaction.This characteristic of the enzyme in which some domains are stable while others appear to be rapidly moving from one conformation state to another before s 'install has not been seen or predicted before,' said Simonovic. "This new information could be used to modulate the way Cas9 treats the targeted DNA and could help design better genome editing tools."
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