In just eight years, CRISPR-Cas9 has become the genome editor best for both basic research and gene therapy. But CRISPR-Cas9 has also created other potentially powerful tools for DNA manipulation that can help fix genetic mutations responsible for hereditary diseases.
Researchers at the University of California, Berkeley, have now obtained the first 3-D structure of one of the most promising of these tools: base editors, which bind to DNA and, instead of cutting, precisely replace a nucleotide. one with another.
First created four years ago, base editors are already being used in attempts to correct single nucleotide mutations in the human genome. The base editors now available can address about 60% of all known genetic diseases ̵1; potentially more than 15,000 inherited disorders – caused by a single nucleotide mutation.
Detailed 3-D structure, reported in the July 31 issue of the journal Science, provides a roadmap for basic tweaking editors to make them more versatile and controllable for use in patients.
“We were able to observe for the first time a grassroots editor in action,” said UC Berdeley fellow postdoctoral fellow Gavin Knott. “We can now understand not only when it works and when it doesn’t, but also design the next generation of base editors to make them even better and more clinically appropriate.”
A base editor is a type of Cas9 fusion protein that employs partially inactivated Cas9 – their cut wounds are disabled so as not to cut a single line of DNA – and an enzyme that, for example, activates or tightens a gene, or modifies areas adjacent DNA. Because the new study reports the first structure of a Cas9 fusion protein, it may help guide the invention of other Cas9-based gene editing tools.
“We actually see for the first time that the base editors behave as two independent modules: You have the Cas9 module that gives you the specificity, and then you have a catalytic module that provides you with the activity,” said Audrone Lapinaite, ex UC Berkeley Postdoctoral Fellow who is now an assistant professor at Arizona State University in Tempe. “The structures we found from this base editor linked to its target really give us a way of thinking about Cas9 fusion proteins, in general, they give us ideas which region of Cas9 is more beneficial for the combination of other proteins. “
Lapinaite and Knott, who recently accepted a position as a research partner at Monash University in Australia, are co-authors of the paper.
Editing one base at a time
In 2012, researchers first demonstrated how to reunite a bacteriological enzyme, Cas9, and turn it into a tool for editing genes in all cell types, from bacteria to humans. The brainchild of UC Berkeley biochemist Jennifer Doudna and her French colleague Emmanuelle Charpentier, CRISPR-Cas9 transformed biological research and brought gene therapy to the clinic for the first time in decades.
Scientists quickly chose Cas9 to produce a large number of tools. Basically a mash-up of protein and RNA, Cas9 accurately targets a specific range of DNA and then precisely creates it, like a pair of scissors. The scissor function can be broken, however, allowing Cas9 to target and bind DNA without cutting. In this way, Cas9 can bind different enzymes to targeted regions of DNA, allowing the enzymes to manipulate genes.
In 2016, Harvard University’s David Liu combined Cas9 with another bacterial protein to allow precise surgical replacement of one nucleotide with another: the first base editor.
While the early adenine base editor was slow, the latest version, called ABE8e, is fast-paced: Complete almost 100% of the intended base editions in 15 minutes. However, ABE8e may be more prone to editing unintended pieces of DNA in a test tube, potentially creating what are known as off-target effects.
The newly revealed structure was obtained using a high-energy imaging technique called cryptoelectronic microscopy (cryoEM). Activity tests have shown why ABE8e is prone to cause further off-target modifications: The deaminase protein dissolved with Cas9 is always active. As Cas9 hops around the nucleus, it sticks and gets rid of hundreds or thousands of DNA segments before it finds its intended target. The attached deaminase, like a loose cannon, doesn’t expect a perfect match and often edits a base before Cas9 gets to rest on its final target.
Knowing how the effector domain and Cas9 are linked can lead to a redesign that makes the enzyme active only when Cas9 has found its target.
“If you really want to design a really specific fusion protein, you need to find a way to make the catalytic domain more part of Cas9, so that it makes sense when Cas9 is on the right target and only then is activated, instead of all the time, “Lapinaite said.
The structure of ABE8e also indicates two specific changes in the deaminase protein that make it work faster than the early version of the base editor, ABE7.10. Those two-point mutations allow the protein to capture DNA more tightly and replace it more efficiently A by G.
“As a structural biologist, I really want to look at a molecule and think about ways to rationally improve it. This structure and the biochemistry that accompanies it really give us that strength,” Knott added. “Now we can make rational predictions for 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 CRISPR gene editing with fewer off-target successes
A. Lapinaite el al., “DNA capture by a CRISPR-Cas9 adenine base editor,” Science (2020). science.sciencemag.org/cgi/doi … 1126 / science.abb1390
Provided by the University of California – Berkeley
Citation: New understanding of CRISPR-Cas9 tool could improve gene editing (2020, 30 July) achieved on 30 July 2020 from https://phys.org/news/2020-07-crispr -cas9-tool-gene.html
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