CRISPR-Cas9’s ability to cut and edit DNA have long been the focus of the genome editing story. But CRISPR-Cas–guided base editors, that specifically correct a common class of base substitutions without cleaving DNA, have recently moved into the spotlight.

In 2016, David Liu, PhD, a chemistry professor affiliated with Harvard University, the Broad Institute, and the Howard Hughes Medical Institute, combined Cas9 with another bacterial protein to allow the precise replacement of one nucleotide with another: the first base editor. Instead of cutting, base editors convert the nucleotides A•T to G•C, or C•G to T•A, in DNA for precision genome editing.

Now, researchers at the University of California (UC), Berkeley, have obtained the first 3D structure of base editors. More specifically, in order to understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), they determined a 3.2-angstrom resolution cryo–EM structure of ABE8e in a substrate-bound state.

The research is published today in Science in the paper, “DNA capture by a CRISPR-Cas9–guided adenine base editor.” The detailed 3D structure provides a roadmap for tweaking base editors to make them more versatile and controllable for use in patients.

“We were able to observe for the first time a base editor in action,” said UC Berkeley postdoctoral fellow Gavin Knott, PhD. “Now we can 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.

CRISPR-Cas9 base editors comprise RNA-guided Cas proteins fused to an enzyme that can deaminate a DNA nucleoside. “We actually see for the first time that base editors behave as two independent modules: You have the Cas9 module that gives you specificity, and then you have a catalytic module that provides you with the activity,” said Audrone Lapinaite, PhD, a former UC Berkeley postdoctoral fellow who is now an assistant professor at Arizona State University in Tempe. “The structures we got of this base editor bound to its target really give us a way to think about Cas9 fusion proteins, in general, giving us ideas which region of Cas9 is more beneficial for fusing other proteins.”

The high resolution structure is of ABE8e bound to DNA in which the target adenine is replaced with an analog designed to trap the catalytic conformation. The structure, together with kinetic data comparing ABE8e to earlier ABEs, explains how ABE8e edits DNA bases and could inform future base-editor design.

Editing one base at a time

While the early adenine base editor was slow, the newest version—ABE8e—is blindingly fast: It completes nearly 100% of intended base edits in 15 minutes. Yet, ABE8e may be more prone to edit unintended pieces of DNA in a test tube, potentially creating off-target effects.

Activity assays showed why ABE8e is prone to create more off-target edits: The deaminase protein fused to Cas9 is always active. As Cas9 hops around the nucleus, it binds and releases hundreds or thousands of DNA segments before it finds its intended target. The attached deaminase doesn’t wait for a perfect match and often edits a base before Cas9 comes to rest on its final target.

The authors wrote that the kinetic and structural data “suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA–like conformation.” They continued, “ABE8e’s accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9.”

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 truly specific fusion protein, you have to find a way to make the catalytic domain more a part of Cas9, so that it would sense when Cas9 is on the correct target and only then get activated, instead of being active all the time,” Lapinaite said.

The structure of ABE8e also pinpoints 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 grip the DNA tighter and more efficiently replace A with G.

“As a structural biologist, I really want to look at a molecule and think about ways to rationally improve it. This structure and accompanying biochemistry really give us that power,” Knott added. “We can now make rational predications for how this system will behave in a cell, because we can see it and predict how it’s going to break or predict ways to make it better.”

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