Directed evolution in mammalian cells

Hendel, Samuel J.; Shoulders, Matthew D.

doi:10.1038/s41592-021-01090-x

Cited by 48 publications

(49 citation statements)

References 104 publications

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“…36 AaRSs evolved in yeast may also be transferrable to other eukaryotes, albeit with potential changes in activity. 37 To the best of our knowledge, all yeast-based campaigns to identify aaRSs for ncAA incorporation have utilized the same set of positive and negative selections. Characterizations of selected aaRSs with advanced reporter systems in our group indicate that the performance of aaRSs available in yeast is highly variable, and the available OTSs cover only a limited range of chemical functionalities.…”

Section: Introductionmentioning

confidence: 99%

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

Stieglitz

2021

Preprint

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Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting (FACS) to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific/polyspecific properties are invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

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Section: Introductionmentioning

confidence: 99%

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

Stieglitz

2021

Preprint

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“…The timing, location, and kinetics of these interactions are critical for the proper function of the proteins. The protein folding, post-translational modifications, localizations and interactions with other proteins are needed for performance in the cell [17].…”

Section: Discussionmentioning

confidence: 99%

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

Mahfouz

Butt

Ramirez

2021

Preprint

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Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP–DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated in the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

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“…More broadly, now that we have validated the proximity dependent split RNAP as a system to select for and evolve molecular glues, this opens up the possibility of implementing this selection scheme with other evolution technologies. For example, implementing the molecular glue selection with the yeast-based Orthorep, mammalian-based mPACE, or VEGAS systems would allow for evolution of molecules that drive the interactions between proteins that cannot be expressed in E. coli, a key limitation of PACE-based methods [70][71][72][73] . As the repertoire of rapid evolutionary technologies expands, so too will our ability to harness evolution to solve problems in synthetic biology and therapeutic design.…”

Section: Discussionmentioning

confidence: 99%

“…In the realm of PPIs, pioneering and robust directed evolution methods such as phage, mRNA, and ribosomal display technologies have generated PPIs with subnanomolar binding affinities [65][66][67] . A range of powerful continuous evolution technologies that reduce the need for human intervention, accelerate evolution, and evolve molecules in unique biological contexts have emerged, including Phage-Assisted Continuous Evolution (PACE), Orthorep, mammalian phage assisted continuous evolution (mPACE), and viral evolution of genetically actuating sequences (VEGAS) [68][69][70][71][72][73] . While powerful, all of these directed evolution approaches are limited by the selection systems that can be deployed, and a robust method to evolve molecules to drive a desired intracellular PPI is lacking.…”

mentioning

confidence: 99%

A system for the evolution of protein-protein interaction inducers

Dewey

Azizi

et al. 2021

Preprint

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Molecules that induce interactions between proteins, often referred to as "molecular glues", are increasingly recognized as important therapeutic modalities and as entry points for rewiring cellular signaling networks. Here, we report a new PACEbased method to rapidly select and evolve molecules that mediate interactions between otherwise non-interacting proteins: rapid evolution of Protein-Protein Interaction Glues (rePPI-G). By leveraging proximity-dependent split RNA polymerase-based biosensors, we developed E. coli-based detection and selection systems that drive gene expression outputs only when interactions between target proteins are induced. We then validated the system using engineered bivalent molecular glues, showing that rePPI-G robustly selects for molecules that induce the target interaction. Proof-of-concept evolutions demonstrated that rePPI-G reduces the "hook" effect of the engineered molecular glues, due at least in part to tuning the interaction affinities of each individual component of the bifunctional molecule. Altogether, this work validates rePPI-G as a continuous, phagebased evolutionary technology for optimizing molecular glues, providing a strategy for developing molecules that reprogram protein-protein interactions.

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Directed evolution in mammalian cells

Cited by 48 publications

References 104 publications

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

A system for the evolution of protein-protein interaction inducers

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