New understanding of CRISPR-Cas9 tool could improve gene editing
Cryo-EM captures CRISPR-Cas9 base editor in action

Date:
July 30, 2020
Source:
University of California - Berkeley
Summary:
Of the CRISPR-Cas9 tools created to date, base editors have gotten
lots of attention because of their seemingly simple editing: they
neatly replace one nucleic acid with another, in many cases all
that should be needed to fix a genetic disease. Scientists have
now determined the structure of the latest base editor as it swaps
out nucleic acids, showing why it can go off target but also how
it can be improved.



FULL STORY ========================================================================== Within a mere eight years, CRISPR-Cas9 has become the go-to genome
editor for both basic research and gene therapy. But CRISPR-Cas9 also
has spawned other potentially powerful DNA manipulation tools that could
help fix genetic mutations responsible for hereditary diseases.


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

First created four years ago, base editors are already being used in
attempts to correct single-nucleotide mutations in the human genome. Base editors now available could address about 60% of all known genetic
diseases -- potentially more than 15,000 inherited disorders -- caused
by a mutation in only one nucleotide.

The detailed 3D structure, reported in the July 31 issue of the journal Science, provides a roadmap for tweaking base editiors 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. "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." A base editor is a type of Cas9 fusion protein
that employs a partially deactivated Cas9 -- its snipping shears are
disabled so that it cuts only one 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 could help guide the invention of myriad other 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 specificity,
and then you have a catalytic module that provides you with the activity,"
said Audrone Lapinaite, 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." Lapinaite and Knott, who recently accepted a position as a research fellow
at Monash University in Australia, are co-first authors of the paper.

Editing one base at a time In 2012, researchers first showed how to
reengineer a bacterial enzyme, Cas9, and turn it into a gene-editing
tool in all types of cells, from bacterial to human. The brainchild
of UC Berkeley biochemist Jennifer Doudna and her French colleague,
Emmanuelle Charpentier, CRISPR-Cas9 has transformed biological research
and brought gene therapy into the clinic for the first time in decades.

Scientists quickly co-opted Cas9 to produce a slew of other
tools. Basically a mash-up of protein and RNA, Cas9 precisely targets
a specific stretch of DNA and then precisely snips it, like a pair of
scissors. The scissors function can be broken, however, allowing Cas9
to target and bind DNA without cutting. In this way, Cas9 can ferry
different enzymes to targeted regions of DNA, allowing the enzymes to manipulate genes.



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

While the early adenine base editor was slow, the newest version, called
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 what are known as
off-target effects.

The newly revealed structure was obtained with a high-powered imaging
technique called cryo-electron microscopy (cryoEM). 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, like a loose cannon,
doesn't wait for a perfect match and often edits a base before Cas9
comes 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 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."

========================================================================== Story Source: Materials provided by
University_of_California_-_Berkeley. Original written by Robert
Sanders. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Audrone Lapinaite, Gavin J. Knott, Cody M. Palumbo, Enrique
Lin-Shiao,
Michelle F. Richter, Kevin T. Zhao, Peter A. Beal, David R. Liu,
Jennifer A. Doudna. DNA capture by a CRISPR-Cas9-guided adenine
base editor.

Science, 2020; 369 (6503): 566 DOI: 10.1126/science.abb1390 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200730141315.htm

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