A new understanding of the CRISPR-Cas9 tool could improve gene editing

In just eight years, CRISPR-Cas9 has become the reference genome publisher for basic research and gene therapy. But CRISPR-Cas9 has also spawned other potentially powerful DNA manipulation tools that could help repair the genetic mutations responsible for inherited diseases.

Researchers at the University of California, Berkeley, have now obtained the first three-dimensional structure from one of the most promising tools: base editors, which bind to DNA and, rather than cut, precisely replace one nucleotide with another.

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

The detailed three-dimensional structure, reported in the July 31 issue of the magazine Science, provides a roadmap for adjusting the basic editors to make them more versatile and controllable for patient use.

“We were able to see a grassroots editor in action for the first time,” said Gavin Knott, a UC Berkeley postdoctoral fellow. “Now we can understand not only when it works and when it doesn’t, but also design the next generation of basic editors to make them even better and more clinically appropriate.”

A base editor is a type of Cas9 fusion protein that employs a partially inactivated Cas9 (its cutting shears are disabled so that it only cuts a strand of DNA) and an enzyme that, for example, activates or silences a gene, or modifies adjacent areas DNA Because the new study reports the first structure of a Cas9 fusion protein, it could help guide the invention of a myriad of other Cas9-based gene editing tools.

“In fact, we see for the first time that base editors behave like two independent modules: it has the Cas9 module that gives it specificity and then it has a catalytic module that gives it 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 from this base publisher coupled with their goal really give us a way of thinking about Cas9 fusion proteins, generally giving us insights into which region of Cas9 is most beneficial for fusing other proteins.”

Lapinaite and Knott, who recently accepted a position as a researcher at Monash University in Australia, are the first authors of the article.

Editing one base at a time

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

Scientists quickly turned to Cas9 to produce a number of other tools. Basically a combination of proteins and RNA, Cas9 precisely targets a specific stretch of DNA and then cuts it precisely, like a pair of scissors. However, the scissor function can be broken, allowing Cas9 to target and bind to uncut DNA. In this way, Cas9 can transport different enzymes to specific regions of DNA, allowing enzymes to manipulate genes.

In 2016, David Liu of Harvard University combined a Cas9 with another bacterial protein to enable the surgically accurate replacement of one nucleotide with another: the first-base publisher.

While the first adenine base editor was slow, the newer version, called ABE8e, is incredibly fast: it completes almost 100% of the planned base editions in 15 minutes. However, ABE8e may be more prone to editing unwanted DNA fragments in a test tube, potentially creating what are known as off-target effects.

The newly revealed structure was obtained with a high power imaging technique called cryoelectron microscopy (cryoEM). Activity assays showed why ABE8e is prone to creating more off-target edits: Cas9-fused protein deaminase is always active. When Cas9 jumps around the nucleus, it binds and releases hundreds or thousands of DNA segments before finding its target. The attached deaminase, like a loose cannon, doesn’t expect a perfect match and often edits a base before Cas9 rests on its ultimate goal.

Knowing how the effector domain and Cas9 are linked can lead to a redesign that activates the enzyme only when Cas9 has found 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 makes sense when Cas9 is on the right target and only then activates, rather than activates I activate all the time, “Lapinaite said.

The structure of ABE8e also points to two specific changes in protein deaminase that make it work faster than the initial version of the base editor, ABE7.10. Those two-point mutations allow the protein to adhere more to DNA and more efficiently replace A with G.

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