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2020-03-18| Technology

Evolved Base Editor Surpasses Predecessors with High Efficiency and Versatility

by Ruchi Jhonsa
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By Ruchi Jhonsa, Ph.D.

 

Base editors are genome editing molecular machines that work by chemically converting and correcting one nucleotide at a time. An improvement on the existing adenine base editors or ABE’s, a team of researchers led by Dr. David Liu at Broad Institute of MIT and Harvard has developed an ABE that can target a large number of genomic locations with higher efficiency. This means that the new base editor can correct many more A-T mutations to G-C in the genome, which were previously inaccessible to its predecessor. This new editor has been named as ABE8e. The findings of this research have been described in the Nature Biotechnology issue published on 16th March.

 

Discovery of the First Base Editor

In November 2013, in an email conversation, Dr. David Liu told his new postdoc Dr. Komor, about a potential project that could transform genome engineering and possibly human therapeutics. He was discussing developing a gene-editing molecular machine that can change a single DNA base at a time. “In principle, converting an A-T base pair to G-C pair could fix up to half of the known pathogenic point mutations in humans,” said Dr. David Liu, core institute member, Richard Merkin Professor, and Director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute.

In 2016, Dr. Komor published the very first base editor that can convert cytosine to uracil on genomic locations by combining cytosine deaminase with a Cas9 protein. At the same time, another student in Dr. Liu’s lab started working on editors that can exchange guanine for adenosine. By repeatedly mutating a sequence of adenine deaminase, she found a protein that could exchange A for G on a single-stranded DNA. Similar to the cytosine base editor, she combined this modified adenosine deaminase with Cas9 and developed an adenosine base editor. Immediately after the release of these two base editors, numerous academic establishments started using them for modifying single bases in the gene sequences and repairing pathogenic point mutations. The potential of this technology led to the launch of Beam Therapeutics in 2017 that went public this year and raised $180 million through its IPO of 1 million shares.

 

Need for New Base Editors

Base editors are primarily made up of inactive CRISPR-Cas9 or –Cas12 enzyme, guided by RNA to locate and bind to its target, and a deaminase enzyme, which does the actual nucleotide conversion. However, base editors have limitations that prevent it from editing anywhere in the genome. This is because, the original base editor was developed to work only with a certain type of Cas9 called SpCas9, which could target the editing machine to only certain locations. When fused with other versions of Cas9 or Cas12 endonucleases, the deaminases worked, albeit with low efficiency.

This led Dr. Liu’s team to modify existing base editors to something that can recognize more target sites via different versions of Cas9 and Cas12 proteins without any loss in efficiency. They used the engineered bacteriophages and bacteria, the system-first developed in his lab to rapidly evolve adenine base editor under laboratory conditions. The system is designed in such a way that the bacteriophage would survive only if adenine base editor acquired mutations that would make it faster and more compatible with the new version of Cas9 and Cas12 proteins. After multiple rounds of laboratory-induced evolution, a new base editor was born called ABE8e.

 

The New and Improved Version

The new base editor, ABE8e was compared for its base editing efficiency, off-target effects and its broader applicability on genomic sites with the existing base editor ABE7.10. It was determined that the new evolved editor could edit DNA at a rate nearly 590 times faster than the original adenine base editor. Jennifer Doudna, one of the CRISPR pioneers and collaborator of the study said, “ABE8e’s speed was like a rocket. Within the first five minutes of our initial assay, it had converted all of the starting material we gave it. The enzyme was remarkably fast.”

But the problem with this highly efficient fast system is that it produces off-target effects at equally high speed. Liu’s team anticipated this problem and made a small change in the base editor domain that significantly reduced the affinity of the base editor for DNA and RNA and increased the dependency on Cas9 for DNA attachment without altering the editing efficiency.

 

Does it Work in Mammalian Cells?

Following the characterization of the altered editor in vitro, the researchers decided to do the final test it on human cells. The team tested the editor’s efficiency on two different genomic sites on BCL11A enhancer, a DNA element that silences fetal hemoglobin gene and two sites on the promoter of HBG genes that transcribe fetal hemoglobin. BCL11A silencer and HBG promoter are attractive targets for treating blood diseases like beta-thalassemia and sickle cell anemia as silencing or activating it respectively would increase levels of fetal hemoglobin in patients whose adult hemoglobin production is compromised.

In comparison to the existing editor, ABE7.10, which could only target 8% of the BCL11A alleles, ABE8e was able to fix 55% of the target allele. This high efficiency was believed to be a result of the high-speed editing of ABE8e. With this feature, ABE8e is now suitable for other versions of Cas9 and Cas12, which engage DNA for short time but cover a vast stretch of genome. Liu and his colleagues are now aiming at correcting pathogenic mutations in animal models of human genetic diseases.

Dr. Liu said, “This editor advances the capabilities of adenine base editing by substantially enhancing its targeting scope, efficiency, and range of applications. We’re thrilled to add ABE8e to the community’s rapidly growing collection of base editing tools. Thus far, ABE8e is working remarkably well. Which is not to say that there won’t be other desired features that we or other labs might want to install in future base editors. But this study and many additional innovations in base editing from other labs around the world demonstrates that the base editing field continues to tailor these molecular machines to offer increasingly powerful and therapeutically relevant properties.”

Related Article: Prime Editing: A New Twist in the CRISPR Tale

References
  1. https://www.broadinstitute.org/news/more-versatile-and-efficient-base-editor-unlocks-new-gene-editing-targets
  2. Richter et al., 2020, Nature Biotechnology
  3. Rees and Liu, 2018, Nat Rev Genet
  4. Gaudelli et al., 2017 Nature
  5. Komor et al., 2016 Nature

 

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