New understanding of CRISPR-Cas9 tool can improve gene editing

The 3D structure of a base editor, which contains the Cas9 protein (white and gray), which binds the DNA target (teal and blue helix), complementary to the RNA guide (purple), and the deaminaminase protein (red and pink). Huh. Which switches one nucleotide to another. Credit: UC Berkeley image by Gavin Knott and Audron Lappinite

Within just eight years, CRISPR-Cas9 has become the go-to genome editor for both basic research and gene therapy. But CRISPR-Cas9 has also produced other potentially powerful DNA manipulation tools that may help to fix genetic mutations responsible for hereditary diseases.

Researchers at the University of California, Berkeley have now obtained one of the most promising of these devices, the first 3-D structure: base editors, which bind DNA and, instead of cutting, replace one nucleotide properly with another.

First created four years ago, base editors are already being used in efforts to correct single-nucleotide mutations in the human genome. Now available base editors can address about 60% of all known genetic diseases – possibly more than 15,000 inherited disorders due to mutations in only one nucleotide.

Detailed 3-D structure, reported in the July 31 issue of the Journal Science, Provides a roadmap for adding Aadhaar editors to make them more versatile and controllable for use in patients.

“We were able to observe for the first time as a base editor in action,” UC Berkeley postdoctoral fellow Gavin Knott said. “Now we can not only understand when it works and when it doesn’t, but by designing the next generation of base editors to make them even better and more medically appropriate.”

A base editor is a type of Cas9 fusion protein that employs partially inactivated Cas9 – its snipping scissors are disabled so that it cuts off only one strand of DNA and an enzyme that activates or silences genes, for example Does, or modifies adjacent areas. Dna Because the first structure of Cas9 fusion protein is reported in the new study, it may help direct the invention of innumerable Cas9-based gene-editing tools.

“We actually see for the first time that base editors behave as two independent modules: you have the Cas9 module that gives you exclusivity, and then you have a catalytic module that gives you activity,” one Former UC Berkeley called Audrone Lapinaite. Postdoctoral fellow who is now an assistant professor at Arizona State University in Tempo. “The base editor’s structures that bind us to its goal actually give us a way to think about the Cas9 fusion protein, in general, giving us an idea of ​​which region of Cas9 is more beneficial for the fusion of other proteins. is.”

Lappinite and Knott, who recently accepted a position as a research fellow at Monash University in Australia, are co-first authors of the paper.

Cryo-EM structure (ABE8e) of the base editor in the function of editing DNA. Credit: Gavin J. Not (PDB ID: 6 VPC)

Editing one base at a time

In 2012, researchers first demonstrated how a bacterial enzyme, Cas9, is reproduced, and is transformed into a gene-editing tool in all types of cells from bacteria to humans. The brainchild of UC Berkeley biochemist Jennifer Dudna and her French colleague, Emmanuelle Chapier, CRISPR-Cas9, has transformed biological research and brought it into a gene therapy clinic for the first time in decades.

Scientists quickly co-opted Cas9 so that other devices could be manufactured. Originally a mash-up of proteins and RNA, Cas9 precisely targets a specific stretch of DNA and then precisely represses it like a pair of scissors. The scissor function can be broken, however, allowing it to target and bind DNA without cutting Cas9. Thus, Cas9 can ferry different enzymes to targeted regions of DNA, allowing enzymes to manipulate genes.

In 2016, David Liu of Harvard University combined a Cas9 with another bacterial protein to allow surgically precise replacement of a nucleotide: first base editor.

While the initial EdinBase base editor was slow, the latest version, called ABE8e, is blinding fast: it accomplishes almost 100% intended base editing in 15 minutes. Nevertheless, ABE8e may be more prone to editing unexpected pieces of DNA in the test tube, potentially creating what is known as the off-target effect.

The newly revealed structure was obtained with a high-power imaging technique called cryo-electron microscopy (cryoim). Activity assays showed why it is prone to make ABE8e a more targeted target: the deaminamin protein fused to Cas9 is always activated. As Cas9 rotates around the nucleus, it binds and releases hundreds or thousands of DNA fragments before finding its intended target. The attached deaminess, like a loose cannon, does not wait for a perfect match and often edits a base before Kas9 arrives at the final goal.

Knowing how the effector domain and Cas9 are interconnected can lead to a redesign that activates the enzyme only when Cas9 has reached its target.

“If you really want to design specific fusion proteins, you have to find a way to make the catalytic domain a part of Cas9, so that it makes sense that when Cas9 is at the right target and only activated, instead of all the time Active, ”Lappinite said.

The structure of ABE8e also indicates two distinct changes in the deaminase protein that make it function faster than the initial version ABE7.10 of the base editor. Those two point mutations allow the protein to cleave DNA and more efficiently replace A with G.

“As a structural biologist, I really want to look at a molecule and rationally think of ways to improve it. This structure and the accompanying biochemistry really give us that power,” Knott said . “Now we can make rational predictions about how this system will behave in a cell, because we can see it and predict how it is going to break or predict ways to improve.”

Secure CRISPR gene editing with fewer off-target hits

more information:
a. Lappinite L. al., “A CRISPR-Cas9-Directed DNA Capture Adenine Base Editor,” Science (2020).… 1126 / science.abb1390

Provided by University of California – Berkeley

Quotes: New understanding of the CRISPR-Cas9 tool can improve gene editing (2020, 30 July) from 30 July 2020

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