In 2013, Caltech chemistry PhD student Alexis Komor interviewed for a postdoctoral position in Liu’s lab. To fulfill a Caltech graduation requirement, Komor had to outline three future research projects. She decided to include one of several project ideas (“white papers”) she’d been discussing with Liu via email, a process he refers to as “mutually guided brainstorming.”
Komor’s initial idea was to evolve a ribonuclease to degrade a specific sequence of RNA. Liu suggested she think about DNA-based editors, in particular the CRISPR-associated nuclease, Cas9. “If you could program a specific A-to-G (for example) change in the human genome,” he emailed her on November 1, 2013, “you could really transform genome engineering and possibly human therapeutics.”
Komor didn’t know much about Cas9, but by the time she arrived at Harvard 10 months later, she was up to speed. One of the first people she met in the lab was Gaudelli, another recently arrived postdoc working on an unrelated project. They were to become fast friends.
Building the first BE molecular machine was a multistep construction challenge.
Komor selected cytidine deaminase, an enzyme that converts cytosine to uracil but works only on single-stranded DNA. She coupled the deaminase to an inactive (“dead”) form of the Cas9 nuclease, which was used to seek out a target sequence in DNA and unspool a short stretch of about five base pairs.
After about six months, she had a prototype BE working, but she faced two major hurdles: to get the BE working in mammalian cells, and then override nature’s DNA repair processes, which would detect the U mismatch and revert the mutation. “[We knew the cell would] change it to something else,” Komor recalls. “The question was, how [could] we force the cell to go in the AT direction rather than just back to a CG base pair?”
Adding a third component—an inhibitor of uracil DNA glycosylase to squelch the natural repair process—helped tip the balance, but not as much as she hoped. Then one day, while talking to a colleague in the break room, she had an epiphany. “It just came to me…we’re working with an endonuclease! We’ve inactivated [Cas9] so all it does is bind [DNA],” but replacing one key amino acid would restore a nickase function that would clip one strand of the double helix.” Komor suddenly saw a way to nick the C-containing strand (leaving the uracil intact) to trigger DNA repair of the C rather than the uracil nucleotide.
When Komor told Liu about her brilliant idea, he started swearing: he’d wanted to start writing up the paper. Komor spent Christmas 2015 at home in southern California revising the manuscript, even foregoing her high school reunion. The paper was eventually accepted and published by Nature in April 2016.
With Komor’s success in engineering the transition of one pyrimidine (C) to another (T), Gaudelli became increasingly distracted about the complementary transition of purines, A to G. After much debate, she abandoned her original project and committed to developing a base editor that would in principle reverse the most common point mutation found in genetic diseases. There was just one problem: there was no naturally occurring adenine deaminase that worked on DNA for Gaudelli to begin building the new BE.
At a crossroads, Gaudelli decided to “double down [and] do the crazy thing!” She broke a golden rule in the Liu lab and set out to evolve a DNA-based ABE. To start, she selected tadA, an adenosine deaminase that works on transfer RNA. Using error-prone PCR, she built gigantic libraries to screen for the desired activity. To her delight, the most significant mutation in the enzyme occurred at the precise residue making contact with the hydrozyl group of the ribose sugar. “[It’s] the one thing that’s different between what I’m trying to make and what I started with,” Gaudelli recalls thinking. She sent a quick slide to Liu—who began swearing again. “Holy —, this is our smoking gun,” he emailed back.
https://blog.addgene.org/single-base-...
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