Cellular Innovation by Rational Design and Evolution

Living cells use circuits of interacting molecules to process signals, remember information, and interact in complex ways with their environments. Our laboratory is interested in creating artificial cellular systems that exceed the design and functional repertoire of nature. Through building these synthetic systems de novo, we are uncovering the design principles of natural cellular circuits and how they carry out specific biological functions. To address the most pressing human diseases, we also use these systems to program useful cellular behaviors in a broader quest to develop next-generation cellular therapeutics.

Our unique approach is to develop, and then creatively integrate, advanced forms of rational design and laboratory evolution to address problems at different levels of cellular organization. First, we develop synthetic biology frameworks to engineer and study the molecular circuits that control gene regulation in eukaryotes. We have made fundamental discoveries involving transcription circuits and epigenetic regulatory systems, and translated these discoveries into platforms for programming therapeutic human cells for personalized medicine. Second, we deepen and expand the functional repertoire of synthetic networks by creating new biomolecule components through advanced continuous evolution methods. To this end, we invented an open-source, automated continuous culture platform called eVOLVER that enables hundreds of microbial populations to be propagated in individually-controlled conditions over long time-scales. We are deploying eVOLVER to enable fundamental eco-evolutionary studies of cellular systems, and to evolve proteins with new and biomedically-useful functions at unprecedented scale.

High-Throughput Continuous Evolution of Compact Cas9 Variants Targeting Single-Nucleotide-Pyrimidine PAMs
Tony P. Huang*, Zachary J. Heins*, Shannon M. Miller, Brandon G. Wong, Pallavi A. Balivada, Tina Wang, Ahmad S. Khalil and David R. Liu
Nature Biotechnology, doi: 10.1038/s41587-022-01410-2 (2022)

High-Performance Multiplex Drug-Gated CAR Circuits
Hui-Shan Li*, Nicole M. Wong*, Elliot Tague, John T. Ngo, Ahmad S. Khalil and Wilson W. Wong
Cancer Cell, 40: 1-12 (2022)

Cooperative Assembly Confers Regulatory Specificity and Long-Term Genetic Circuit Stability
Meghan D. J. Bragdon*, Nikit Patel*, James Chuang, Ethan Levien, Caleb J. Bashor* and Ahmad S. Khalil*
bioRxiv, doi: 10.1101/2022.05.22.492993

In Vivo Hypermutation and Continuous Evolution
Rosana S. Molina, Gordon Rix, Amanuella A. Mengiste, Beatriz Alvarez, Daeje Seo, Haiqi Chen, Juan E. Hurtado, Qiong Zhang, Jorge Donato Garcia-Garcia, Zachary J. Heins, Patrick J. Almhjell, Frances H. Arnold, Ahmad S. Khalil, Andrew D. Hanson, John E. Dueber, David V. Schaffer, Fei Chen, Seokhee Kim, Luis Angel Fernandez, Matthew D. Shoulders, and Chang C. Liu
Nature Reviews Methods Primer, 2: 36 (2022)

Modular Design of Synthetic Receptors for Programmed Gene Regulation in Cell Therapies
Iowis Zhu, Raymond Liu, Julie M. Garcia, Axel Hyrenius-Wittsten, Dan I. Piraner, Josef Alavi, Divya V. Israni, Bin Liu, Ahmad S. Khalil and Kole T. Roybal
Cell, 185: 1431-1443 (2022)

Once Cell, Many Fates (Perspective)
Colin Kunze and Ahmad S. Khalil
Science, 375: 262-263 (2022)

Here to Stay: Writing Lasting Epigenetic Memories (Preview)
Hagar F. Moussa*, James F. Angstman* and Ahmad S. Khalil
Cell, 184: 2281-2283 (2021)

Clinically-Driven Design of Synthetic Gene Regulatory Programs in Human Cells
Divya V. Israni*, Hui-Shan Li*, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong and Ahmad S. Khalil
bioRxiv, doi: 10.1101/2021.02.22.432371

Transcriptional Kinetic Synergy: A Complex Landscape Revealed by Integrating Modelling and Synthetic Biology
Rosa Martinez-Corral, Minhee Park, Kelly Biette, Dhana Friedrich, Clarrissa Scholes, Ahmad S. Khalil, Jeremy Gunawardena and Angela H. DePace
bioRxiv, doi: 10.1101/2020.08.31.276261

Environmental Fluctuations Reshape an Unexpected Diversity-Disturbance Relationship in a Microbial Community
Christopher P. Mancuso, Hyunseok Lee, Clare I. Abreu, Jeff Gore and Ahmad S. Khalil
eLife, 10: e67175 (2021)

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