All Roads Lead to Genome Editing

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Shondra Pruett-Miller, a biochemist at St. Jude Children’s Research Hospital, found her calling during her first year of graduate school at the University of Texas, Southwestern. In 2003, during the departmental faculty research talks, she watched with excitement as Matthew Porteus, now a biochemist at Stanford University, presented his research on zinc finger nucleases (ZFN) as a tool for inducing double-strand break repair for gene editing, with possible gene therapy applications in genetic diseases of the blood.1 “I fell in love with it,” said Pruett-Miller, who later joined Porteus’s team as a graduate student. “I thought, ‘wow, the power of this technology—to be able to go in and make precise modifications—it’s going to change the world!’” She added, “I remember we were like, ‘man, it would be so cool if we could cure sickle cell disease.”

I still credit the success of my lab, now 20 years later, in large part to the culture she created in the lab.
– Matthew Porteus, Stanford University

ZFN were one of the first tools for targeted gene repair, but designing them to cut in a precise spot in the genome was difficult.2 Pruett-Miller sought a protein engineering solution to coax ZFN to cut at a specific location and eventually designed a pair of ZFN to cut green fluorescent protein (GFP).3 “It sounds really sad now, but it was really hard, and it took a lot of time to do that,” said Pruett-Miller.

This occurred during the pre-CRISPR era, and only a handful of companies and academic labs worked with engineered nucleases for genome editing. “Shondra is really one of the pioneers,” said Porteus. Several years before researchers started to explore ways to shorten the half-life of Cas9 to improve the safety of CRISPR-based therapies, Pruett-Miller altered the half-life of ZFN to reduce toxicity.4,5 “She was part of laying that foundational knowledge so that when CRISPR was discovered, everyone sort of knew what to do with it,” Porteus added.

As she cultivated her experimental expertise, Pruett-Miller’s other attributes started to shine. Porteus recalled her leadership skills and the open and collaborative environment she fostered. “I still credit the success of my lab, now 20 years later, in large part to the culture she created in the lab,” said Porteus. 

At the end of her studies, Pruett-Miller arrived at a fork in the road: Should she take the traditional path and become a postdoctoral researcher in an academic lab or embark on a new opportunity in industry? She chose industry. “We were a little disappointed that she went to Sigma because we thought her destiny was to run her own lab,” said Porteus. 

Others expressed sharper opinions. “One person actually told me it’s career suicide to not do a postdoc, and I was like ‘Oh, my gosh, what am I doing?’” said Pruett-Miller.

Not all who wander are lost

Any creeping doubts that Pruett-Miller had soon vanished as she embarked on a new journey that allowed her other scientific skills to flourish. “I’m a good communicator, and that has led me to get to positions of leadership that I might not have gotten had I gone a different path,” she said.

In 2008, she moved to St. Louis for a job at Sigma Aldrich as a senior research and development scientist in their ZFN research team. Starting with just a single pair of ZFN in graduate school, she now had access to any ZFN she wanted. “We were able to do some of the pioneering work in the field,” said Pruett-Miller. “We had the keys to the car where we could actually start driving it forward.” 

She loved working on cutting-edge gene-editing technology, but two and a half years into the position, another unique opportunity presented itself. She joined Sigma’s customer education group, which took her around the world teaching three-day workshops on ZFN technologies. 

Sigma’s marketing and sales teams took notice of her communication skills and offered her yet another position as a field application scientist. “I never would have thought I would have done sales when I was in graduate school,” she said. But the role married her favorite things: teaching, helping scientists plan and troubleshoot experiments, and of course, genome editing.

Nearly five years after joining Sigma, Pruett-Miller saw that the genome editing landscape was rapidly evolving, so, once again, she hoisted the sails to embark on a new journey.2 

A labor of love

Pruett-Miller in a white coat looking at a flask under a microscope.

Shondra Pruett-Miller’s career path prepared her for leadership and management roles. Now, she works closely with her team members to ensure that they find the paths that are right for them. 

St. Jude Children’s Research Hospital 

Pruett-Miller didn’t travel far. She went across town to Washington University in St. Louis, where she pitched an idea for a shared resource that provided genome editing services for researchers. Jeff Milbrandt, a geneticist at Washington University in St. Louis who previously started a genome sequencing core, had his sights set on a similar endeavor for genome engineering. Pruett-Miller’s proposal fit the bill.

Many institutions had core facilities for sequencing, but validating genetic findings was labor-intensive. Pruett-Miller proposed a facility that could provide scientists with the tools needed for robust functional studies. “There was really nobody doing cell line engineering as a service inside of academia, and there’re still very few groups that do that because it’s—I would say—a labor of love,” said Pruett-Miller. 

On her first day on the job, she faced the stark reality of starting from the ground up. She found herself alone in an empty lab, surrounded by ceiling tiles piled high where her lab equipment belonged. Undeterred, she got to work, and soon enough, things fell into place and her team grew. They started working with ZFN and gene editing newcomers transcription activator-like effector nucleases (TALEN), but only six months into the position, CRISPR-Cas9 crashed onto the scene. 6,7 With excitement, she learned the ins and outs of these new technologies and how they could benefit her research community. 

There are people who are basically cured of sickle cell disease walking around because of genome editing and CRISPR-Cas9. Sickle cell disease is just the starting point.
-Shondra Pruett-Miller, St. Jude Children’s Research Hospital

“Shondra did a great job in the startup phase, not unlike you would see in a startup company [where] you have a leader who really is passionate about it, who spends a lot of their time at it, who develops and mentors people to do exciting work,” said Milbrandt. 

Washington University in St. Louis wasn’t the only institution that saw the value of a shared gene editing resource. In 2016, St. Jude Children’s Research Hospital invited Pruett-Miller to establish a similar facility on their campus, but she was happy and proud of the work she had been doing at Washington University in St. Louis. Plus, she was six months pregnant with her second child. “But it’s hard to come to St. Jude’s campus and see the resources and the mission of St. Jude and not be inspired by what they’re doing here,” said Pruett-Miller, who received her offer letter two weeks postpartum and moved with her family nearly 300 miles down the Mississippi River to Memphis, Tennessee. 

Holding true to her bench to bedside mission, her time is now divided between directing the Center for Advanced Genome Engineering, which provides gene editing services to St. Jude Children’s Research Hospital researchers, and her own research developing gene editing therapies for sickle cell disease.8,9 “There are people who are basically cured of sickle cell disease walking around because of genome editing and CRISPR-Cas9,” said Pruett-Miller. “Sickle cell disease is just the starting point.”

Although Pruett-Miller has traveled down many career paths, she remained on course to achieving her scientific goals: translating genome editing to the clinic and making the technology more accessible to the scientific community. “My role now is to shape the next generation of leaders,” said Pruett-Miller. 

References

  1. Porteus MH, Carroll D. Gene targeting using zinc finger nucleases. Nat Biotechnol. 2005;23(8):967-973.
  2. Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
  3. Zou J, et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell. 2009;5(1):97-110.
  4. Yang S, et al. Shortening the half-life of Cas9 maintains its gene editing ability and reduces neuronal toxicity. Cell Rep. 2018;25(10):2653-2659.
  5. Pruett-Miller SM, et al. Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels. PLoS Genet. 2009;5(2):e1000376.
  6. Miller JC, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29(2):142-148.
  7. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
  8. Newby GA, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature. 2021;595(7866):395-302.
  9. Mayuranathan T, et al. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet. 2023;55(7):1210-1220.
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