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- Title
Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis.
- Authors
Jewel, Delilah; Kelemen, Rachel E.; Huang, Rachel L.; Zhu, Zeyu; Sundaresh, Bharathi; Malley, Kaitlin; Pham, Quan; Loynd, Conor; Huang, Zeyi; van Opijnen, Tim; Chatterjee, Abhishek
- Abstract
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.
- Subjects
TRANSFER RNA; CELLULAR evolution; AMINO acids; MUTAGENESIS; CONCURRENT engineering; LIBRARY design &; construction
- Publication
Angewandte Chemie, 2024, Vol 136, Issue 9, p1
- ISSN
0044-8249
- Publication type
Article
- DOI
10.1002/ange.202316428