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

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.

tRNA engineering strategies for genetic code expansion

“…Recently, researchers have employed a virus-mediated gene delivery technique to conduct directed evolution of tRNA within animal cells called virus-assisted directed evolution strategy (VADER) ( Jewel et al, 2023 ; Jewel et al, 2024 ). They employed a selection system diverging from the commonly used positive/negative selection method.…”

Suppressor tRNAs at the interface of genetic code expansion and medicine

“…While bacterial platforms remain the primary avenue for tRNA engineering, recent work established the virus-assisted directed evolution of tRNA (VADER) (Jewel et al, 2023;Jewel et al, 2024). VADER facilitates the screening of >60,000 unique tRNA variants in human cultured cells, offering a novel avenue for rapid human sup-tRNA discovery.…”

Tuning tRNAs for improved translation

“…This emphasizes that the protein synthesis machinery differs between hosts, and tRNA sequences improved in one host cannot necessarily be transferred to others. Follow-up research identified a tRNA Pyl variant, PyOtR, with markedly improved activity over A2.1 in mammalian cells ( Jewel et al, 2024 ). PyOtR contained a similarly GC-rich acceptor stem, with a bias for 5′ G residues, but also demonstrated an essential G5:U68 wobble pair—usually detrimental in tRNA acceptor stems ( Santoro et al, 2003 ).…”