Within just eight years, CRISPR-Cas9 has become the genome editor for both basic research and gene therapy. But CRISPR-Cas9 has also spawned other potentially powerful DNA manipulation tools that could help regulate the genetic mutations responsible for inherited diseases.
Researchers at the University of California, Berkeley, have now taken the first 3-D structure of one of the most promising of these tools: basic editors, which bind to DNA and, instead of cutting, exactly replace one nucleotide with another.
First created four years ago, database editors are already being used in attempts to correct single-nucleotide mutations in the human genome. Basic editors now available can address about 60% of all known genetic diseases ̵1; potentially more than 15,000 inherited disorders – caused by a mutation in just one nucleotide.
Detailed 3-D structure, reported in the July 31 issue of the diary science, provides a guide for editing database editors to make them more comprehensive and controllable for patients to use.
“We were able to observe for the first time a basic editor in action,” said UC Berkeley postdoctoral fellow Gavin Knott. “Now we can understand not only when it works and when it doesn’t, but also create the next generation of database editors to make them even better and more clinically fit.”
A basic editor is a type of Cas9 fusion protein that uses a partially deactivated part of Cas9 ars its deactivated scissors are incapable of cutting a single strand of DNA – and an enzyme that, for example, activates or silences a gene, or modifies adjacent areas of DNA. Because the new study reports the first structure of a Cas9 fusion protein, it may help guide the invention of a number of other tools for editing the Cas9 gene.
“We actually see for the first time that base editors behave like two independent modules: You have the Cas9 module that gives you the specification, and then you have a catalytic module that provides you with activity,” said Audrone Lapinaite, former UC Berkeley is a postdoctoral fellow who is now an assistant professor at Arizona State University in Tempe. “The structures we have for this basic editor are bound to its objective, it really gives us a way to think about Cas9 fusion proteins, in general, giving us ideas of which Cas9 region is most useful for fusion. of other proteins. “
Lapinaite and Knott, who recently accepted a position as a researcher at Monash University in Australia, are the first authors of the paper.
Editing one database at a time
In 2012, researchers showed for the first time how to regenerate a bacterial enzyme, Cas9, and turn it into a tool for editing genes in all cell types, from bacterial to human. The mind for UC Berkeley biochemist Jennifer Doudna and her French colleague Emmanuelle Charpentier, CRISPR-Cas9 has transformed biological research and brought gene therapy to the clinic for the first time in decades.
Scientists quickly decided to choose Cas9 to produce other tools. Essentially a mixture of protein and RNA, Cas9 precisely targets a specific stretch of DNA and then precisely slides it, like a pair of scissors. The function of the scissors can be disrupted, however, by allowing Cas9 to target and bind DNA without cutting. In this way, Cas9 can transport different enzymes to target regions of DNA, allowing enzymes to manipulate genes.
In 2016, Harvard University David Liu combined a Cas9 with another bacterial protein to allow the correct surgical replacement of one nucleotide with another: the first base editor.
While the early adenine base editor was slow, the newer version, called ABE8e, is very fast: It completes almost 100% of the targeted basic edits in 15 minutes. However, ABE8e may be more prone to modify unwanted pieces of DNA in a test tube, potentially creating what are known as non-intentional effects.
The newly discovered structure is taken up with a high-power imaging technique called cryo-electron microscopy (CryoEM). Activity analyzes showed why ABE8e tends to create more off-target edits: The deaminase protein fused to Cas9 is always active. As Cas9 clumps around the nucleus, it binds and releases hundreds or thousands of segments of DNA before it finds its intended target. The attached Deaminaza, like a loose ball, does not expect a perfect match and often edits a base before Cas9 comes to rest on his ultimate goal.
Knowing how the effector domain and Cas9 relate can lead to a redesign that makes that enzyme active only when Cas9 has found its target.
“If you really want to create specific fusion protein, you have to find a way to make the catalytic field more of a part of Cas9, so that it makes sense when Cas9 is at the right target and only then get active, instead of being active all the time, “Lapinaite said.
The structure of ABE8e also shows two specific changes in the deaminase protein that make it function faster than the earlier version of the base editor, ABE7.10. These colon mutations allow the protein to capture DNA more closely and more efficiently replace A with G.
“As a structural biologist, I really want to look at a molecule and think of ways to improve it rationally. This structure and the accompanying biochemistry really give us that power,” Knott added. “Now we can make rational predictions about how this system will behave in a cell, because we can see it and predict how it will break down or predict ways to make it better.”
Safer editing of CRISPR genes with fewer off-target strokes
A. Lapinaite el al., “DNA capture by an adenine base editor run CRISPR-Cas9,” science (2020). science.sciencemag.org/cgi/doi… 1126 / science.abb1390
Provided by the University of California – Berkeley
citation: New understanding of CRISPR-Cas9 tool can improve gene editing (2020, July 30) retrieved July 30, 2020 from https://phys.org/news/2020-07-crispr-cas9-tool-gene. html
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