We selected amber stop code TAG for the following reasons: (1) Previously published papers reported that recoded E. Redesigning and de novo synthesis of yeast genomes project was implemented by the SC 2.0 consortium team, and UAG to UAA recoding in their design 9, 17, 18.īuilding on this previous work, we set out to explore the feasibility of genome-wide TAG to TAA replacement in human cells. In addition to E.coli, 1557 synonymous leucine codons were replaced across 176 genes in Salmonella typhimurium using SIRCAS 16. Deletion of the tRNAs charging the removed serine codons and release factor 1 conferred resistance from a cocktail of viruses, and the blank codons were reassigned to enable the efficient synthesis of proteins containing three distinct nonstandard amino acids in SYN61 3. Parallel efforts have resulted in the complete recoding and assembly of a 61-codon E. Recently, our lab has made over 62,214 changes by synthesizing and assembling a 3.97-megabase, 57-codon E. In another effort, rewriting of 13 sense codons across a set of ribosomal genes 12 and 123 instances of two rare Arginine codons were synonymously replaced 13. This recoding scheme decreased transduction by 4 different bacteriophages (λ, M13, P1, MS2) that infect E. coli with all UAG to UAA replacements and deleted RF1 that enables the termination of translation for UAG and UAA 11. Virus resistance was subsequently tested in engineered E.
Our lab first achieved genome-wide recoding where 314 instances of the UAG stop codon were replaced with UAA in E. Human recoding is also the pilot project of GP-write, which was founded to evolve the “reading” goals of the Human Genome Project into “writing” the next generation genomes 10. Here, we propose human genome recoding to generate virus-resistant cell lines by converting stop codon TAG to TAA, and replacing the endogenous eukaryotic release factor 1 (eRF1) with engineered eRF1 variants (Supplementary Fig. Then, recoding has also been subsequently extended to yeast genome 9, but its application in the human genome has not been reported so far. coli by replacing two sense codons with their synonymous codons, and deleting the corresponding transfer RNA (tRNA) 3. Recently, recoding was implemented genome-wide in E. Recoding was first established in prokaryotes through substitution of the TAG stop codon with TAA and deletion of release factor 1 (RF1) 2, 8.
Recoding confers virus resistance 2, 3, 4, 5, and can also be repurposed to assign the “blank” codons new functions including nonstandard amino acid incorporation 2 and biocontainment 6, 7. Genome recoding is a powerful tool to understand and enhance the genomic function of organisms by genetic engineering. The genetic code is degenerate, assigning 61 triplet codons to 20 naturally occurring amino acids in addition to 3 triplets coding for a stop signal, and 18 of the 20 amino acids are encoded by more than one synonymous codon 1.
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