More versatile and efficient base editor unlocks new gene-editing targets

A team led by researchers at the Broad Institute of MIT and Harvard has designed a new base editing tool that can fix mutations in the genome much more efficiently and at many more target sites compared to its predecessor, unlocking access to correct more genetic variants associated with human diseases.

Base editing is a genome editing technique that chemically transforms one nucleotide into another. The new editor — called ABE8e — can convert A•T base pairs to G•C far faster than the original editor, as measured by collaborators at University of California, Berkeley. ABE8e also edited therapeutic targets in human cells with up to six-fold increased efficiency, and showed a much wider range of compatibility with different CRISPR proteins needed to access targets throughout the genome.

“In principle, converting an A•T base pair to a G•C pair could fix up to half of known pathogenic point mutations in humans,” said senior author David Liu, core institute member, Richard Merkin Professor, and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute. “ABE8e now provides a powerful engine to access more of those mutations — allowing us to perform high-efficiency editing at a broader range of sites in the human genome.” Liu is also professor of chemistry and chemical biology at Harvard, and investigator at the Howard Hughes Medical Institute.

The work is described today in Nature Biotechnology, with postdoctoral scholar Michelle Richter and graduate student Kevin Zhao in Liu’s lab as co-first authors.

Overcoming base editing hurdles

Base editors are primarily constructed with a “deactivated” CRISPR-Cas9 or -Cas12 enzyme, guided by an RNA to locate and bind to its target, and a deaminase protein, which performs the actual nucleotide conversion. However, base editors have limitations on where they can edit. Different versions of the Cas9 and Cas12 enzymes require that a particular stretch of nucleotides, called a “PAM sequence,” exist nearby in order to recognize their targets.

“Base editing relies on having a big ‘bookshelf’ of different Cas variants that you can use to park the editor at different sites in a genome,” explained Liu. “Each different Cas variant will allow you access to different locations.”

The original adenine base editor was designed to work with a version of Cas9 called SpCas9. As the research team explored other options for building these editors, they discovered that the editing power of the deaminase protein was often significantly decreased when coupled with other Cas proteins.

The authors turned to a protein-evolution system to generate new versions of the adenine base editor, ones that could make targeted edits in partnership with many more Cas9 and Cas12 variants. In an ecosystem of engineered bacteriophages and bacteria, this system — first developed in Liu’s lab at Harvard in 2011 to speed up laboratory protein evolution — allows phages to survive and evolve only if they encode proteins with desired traits. For this study, the phages could replicate only if the adenine base editor acquired mutations that made it faster and more compatible with other Cas9 and Cas12 proteins.

After many generations of continuous laboratory evolution, the system generated a new base editor variant that the team called ABE8e.

Understanding ABE8e

To characterize ABE8e more in-depth, Liu and his colleagues shared the base editor with a team led by Jennifer Doudna, professor of molecular and cell biology and of chemistry at University of California, Berkeley, and investigator at the Howard Hughes Medical Institute.

Using biochemical assays in a test tube, Doudna’s team showed that ABE8e could edit DNA at a rate nearly 600 times faster than the original adenine base editor.

“ABE8e’s speed was like a rocket,” said Doudna. “Within the first five minutes of our initial assay, it had converted all of the starting material we gave it. The enzyme was just remarkably fast.”

Because of ABE8e’s faster editing power, Liu’s team also expected it to make more mistakes, or “off-target” edits — so they incorporated additional tweaks to the protein to successfully reduce off-target DNA and RNA editing activity, without impairing its “rocket”-like speed. 

When using eight different Cas9 and Cas12 variants for DNA targeting, ABE8e successfully and efficiently edited its targets in human cells. These new capabilities enabled the team to access and edit a gene called BCL11A, involved in silencing fetal hemoglobin — an attractive target for therapies that could treat blood diseases including sickle-cell anemia. For example, editing DNA to turn fetal hemoglobin production back on could restore blood function in patients whose adult hemoglobin genes are mutated.

In a head-to-head comparison, the original adenine base editor could only edit BCL11A in approximately eight percent of human cells. ABE8e successfully edited more than 50 percent.

The team suspects that the high-speed editing of ABE8e is responsible for this success. SpCas9, the targeting component of the original adenine base editor, may bind DNA more robustly than other Cas protein variants, giving the deaminase time to complete its chemical reactions. ABE8e’s new level of speed now enables it to work with natural and engineered Cas variants that engage DNA for shorter periods of time. The team is working to further characterize ABE8e and illuminate the molecular basis of these improved features.

“This editor advances the capabilities of adenine base editing by substantially enhancing its targeting scope, efficiency, and range of applications,” said Liu. “We’re thrilled to add ABE8e to the community’s rapidly growing collection of base editing tools.”

This study was supported in part by the NIH (U01 AI142756, RM1 HG009490, R01 EB022376, and R35 GM118062), St. Jude Collaborative Research Consortium, the Bill and Melinda Gates Foundation, and the Howard Hughes Medical Institute.