In just eight years, CRISPR-Cas9 has become the genome editor for basic research and gene therapy. However, CRISPR-Cas9 has also developed other potentially powerful DNA manipulation tools that could help correct genetic mutations responsible for inherited diseases.
Researchers at the University of California, Berkeley, have now obtained the first three-dimensional structure of one of the most promising tools: base editors that bind to DNA and, instead of cutting, precisely replace one nucleotide with another.
Base editors, first created four years ago, are already being used in attempts to correct single nucleotide mutations in the human genome. Currently available base editors can address about 60% of all known genetic diseases ̵1; potentially more than 15,000 inherited disorders – caused by mutations in just one nucleotide.
Detailed three-dimensional structure given in the July 31 issue of the magazine science, provides a plan for tweaking basic editors to make them more versatile and controllable for use by patients.
“We were able to observe the base editor in action for the first time,” said Gavin Knott, UC Berkeley’s postdoctoral colleague. “Now we can understand not only when it works and when it doesn’t, but also to design the next generation of foundation editors to make them even better and more clinically appropriate.”
A base editor is a type of Cas9 fusion protein that utilizes partially inactivated Cas9 – its shears are inactivated by cleaving only one strand of DNA – and an enzyme that, for example, activates or silences a gene or modifies adjacent regions. DNA. Because the new study presents the first structure of the Cas9 fusion protein, it could help guide the invention of countless other Cas9-based gene editing tools.
“In fact, for the first time, we see database editors behaving like two independent modules: You have a Cas9 module that gives you specificity, and then you have a catalytic module that gives you activity,” said Audrone Lapinaite, a former UC Berkeley. a postdoctoral fellow who is now an assistant professor at Arizona State University in Tempe. “The structures we got from this target-bound base editor really give us a way to think of Cas9 fusion proteins, in general, giving us an idea of which Cas9 region is more suitable for fusion of other proteins.”
Lapinaite and Knott, who recently accepted the position of researcher at Monash University in Australia, are co-authors of this article.
Modifications of one base at a time
In 2012, researchers first showed how to reengineer the bacterial enzyme Cas9 and turn it into a tool for gene modification in all cell types, from bacterial to human. UC Berkeley brain biologist 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 co-opted Cas9 to make other tools killed. Essentially a mash-up of proteins and RNA, Cas9 precisely targets a specific stretch of DNA and then precisely cuts it off as scissors. However, scissor function can be disrupted, allowing Cas9 to target and bind DNA without cutting. In this way, Cas9 can transport various enzymes to target regions of DNA, allowing enzymes to manipulate genes.
In 2016, David Liu of Harvard University combined Cas9 with another bacterial protein to allow surgically accurate replacement of one nucleotide with another: the first base editor.
While the initial adenine base editor was slow, the latest version called ABE8e is blindingly fast: It completes almost 100% of the planned basic edits in 15 minutes. Nevertheless, ABE8e may be more prone to modify unintentional parts of DNA in a test tube, potentially creating so-called off-target effects.
The newly discovered structure was obtained by a high-performance imaging technique called cryo-electron microscopy (cryoEM). Activity assays have shown why ABE8e is prone to form more off-target modifications: The deaminase protein fused to Cas9 is always active. When Cas9 orbits the nucleus, it binds and releases hundreds or thousands of segments of DNA before it finds its intended target. The attached deaminase, like the free cannon, does not wait for a perfect match and often adjusts the base before Cas9 stops at 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 finds its target.
“If you really want to design a truly specific fusion protein, you have to find a way to make the catalytic domain more part of Cas9, so it would make sense when Cas9 is on the right target and only then activated, instead of still being active,” he said. Lapinaite.
The structure of ABE8e also identifies two specific changes in the deaminase protein that make it run faster than an early version of the basic editor, ABE7.10. These two point mutations allow the protein to grasp DNA more firmly and more effectively 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 this power, “added Knott. “Now we can rationally predict how this system will behave in the cell, because we can see it and predict how it will break or predict ways to improve it.”
Safer editing of CRISPR genes with fewer off-target hits
A. Lapinaite et al., “DNA capture using the adenovirus base editor guided by CRISPR-Cas9”, science (2020). science.sciencemag.org/cgi/doi… 1126 / science.abb1390
Provides University of California – Berkeley
Citations: A new understanding of CRISPR-Cas9 could improve gene modifications (2020, July 30) obtained on July 30, 2020 from https://phys.org/news/2020-07-crispr-cas9-tool-gene.html
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