Virus-assisted directed evolution of biomolecules

Jewel, Delilah; Pham, Quan Thanh; Chatterjee, Abhishek

doi:10.1016/j.cbpa.2023.102375

Cited by 8 publications

(6 citation statements)

References 57 publications

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“…Viruses provide a distinctive avenue for designing rapid laboratory evolution experiments, capitalizing on their inherent capacity to evolve at a much faster pace than many living organisms [105]. This accelerated evolution is facilitated by their smaller genome size, which tolerates a high frequency of mutations and a rapid rate of replication.…”

Section: Virus-assisted Mutagenesismentioning

confidence: 99%

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Gali,

Tee,

Wong

2024

SynBio

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Genetic diversity is the foundation of evolutionary resilience, adaptive potential, and the flourishing vitality of living organisms, serving as the cornerstone for robust ecosystems and the continuous evolution of life on Earth. The landscape of directed evolution, a powerful biotechnological tool inspired by natural evolutionary processes, has undergone a transformative shift propelled by innovative strategies for generating genetic diversity. This shift is fuelled by several factors, encompassing the utilization of advanced toolkits like CRISPR-Cas and base editors, the enhanced comprehension of biological mechanisms, cost-effective custom oligo pool synthesis, and the seamless integration of artificial intelligence and automation. This comprehensive review looks into the myriad of methodologies employed for constructing gene libraries, both in vitro and in vivo, categorized into three major classes: random mutagenesis, focused mutagenesis, and DNA recombination. The objectives of this review are threefold: firstly, to present a panoramic overview of recent advances in genetic diversity creation; secondly, to inspire novel ideas for further innovation in genetic diversity generation; and thirdly, to provide a valuable resource for individuals entering the field of directed evolution.

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Section: Virus-assisted Mutagenesismentioning

confidence: 99%

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Gali,

Tee,

Wong

2024

SynBio

View full text Add to dashboard Cite

show abstract

“…Viruses provide a distinctive avenue for designing rapid laboratory evolution experiments, capitalizing on their inherent capacity to evolve at a much faster pace than many living organisms [77]. This accelerated evolution is facilitated by their smaller genome size, which tolerates a high frequency of mutations, and a rapid rate of replication.…”

Section: Virus-assisted Mutagenesismentioning

confidence: 99%

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Gali,

Tee,

Wong

2023

Preprint

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Genetic diversity is the foundation of evolutionary resilience, adaptive potential, and the flourishing vitality of living organisms, serving as the cornerstone for robust ecosystems and the continuous evolution of life on Earth. The landscape of directed evolution, a powerful biotechnological tool inspired by natural evolutionary processes, has undergone a transformative shift propelled by innovative strategies for generating genetic diversity. This shift is fuelled by several factors, encompassing the utilization of advanced toolkits like CRISPR-Cas and base editors, enhanced comprehension of biological mechanisms, cost-effective custom oligo pool synthesis, and the seamless integration of artificial intelligence and automation. This comprehensive review looks into the myriad methodologies employed for constructing gene libraries, both in vitro and in vivo, categorized into three major classes: random mutagenesis, focused mutagenesis, and DNA recombination. The objectives of this review are threefold: firstly, to present a panoramic overview of recent advances in genetic diversity creation, secondly, to inspire novel ideas for further innovation in the genetic diversity generation, and thirdly, to provide a valuable resource for individuals entering the field of directed evolution.

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“…Although semi‐rational approaches have been employed with some success to improve the activity of orthogonal suppressor tRNAs in mammalian cells, such approaches are difficult to generalize [6] . Recently, we reported a novel platform for virus‐assisted directed evolution of tRNAs (VADER) in mammalian cells (Figure 1a), which employs a double‐sieve selection scheme to enrich orthogonal tRNA mutants with higher activity from synthetic libraries [2a,d] . The VADER protocol subjects adeno‐associated virus (AAV) particles encoding a suppressor tRNA library to selective replication in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis

Jewel,

Kelemen,

Huang

et al. 2024

Angew Chem Int Ed

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Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNAPyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.

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Virus-assisted directed evolution of biomolecules

Cited by 8 publications

References 57 publications

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Crafting Genetic Diversity: Unlocking the Potential of Protein Evolution

Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis

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