Church's lab frees 14 codons by rewriting the ribosome's lock instead of the genome

Using AGENTEX, the authors evaluated two genetic codes alongside the standard genetic code, with non-standard amino acid incorporation and reassignment of up to three codons.

The approach uses pools of otRNAs to specify unique genetic codes using ribosomes with altered large subunit.

According to phys.org, AGENTEX makes 34 codons customizable, allowing researchers to design proteins using up to 34 amino acids rather than the 20 naturally occurring ones, in test tubes without touching an organism's natural DNA.

Church said: "It took us a decade per new amino acid added to the code, so this remarkably opens the door to 34 at once and with almost none of the usual collateral damage to the genome."

Progress in codon reprogramming has been slow and fraught, including because codon reprogramming hampers other normal cell functions and because genetically engineered organisms need to be kept safely walled off from natural life.

The 34-codon figure is arithmetically the 20 canonical amino acids plus the 14 codons that compression liberates.

Compressing the 64-codon near-universal genetic code to only 20 sense codons, one start codon and one stop codon liberates 14 codons for non-standard building blocks.

The standard genetic code uses 64 codons to encode 20 canonical amino acids across domains of life.

Lysate cell-free systems are produced from crude cell extracts and retain both translational machinery and enzymes for RNA and post-translational modifications, support native tRNA modifications and scale for bioproduction, but contain the complete tRNA set of the host, which can exhibit crosstalk with reassigned sense codons and obscure synthetic genetic code designs.

PURExpress (PURE) uses individually purified components combined in defined ratios; proof-of-concept genetic code expansion studies and directed evolution have largely been performed in PURE, although PURE is less suitable for preparative-scale bioproduction.

Genetic code expansion has enabled the introduction of more than 400 non-standard amino acids into proteins in living cells.

Genetic code expansion to date has used engineered ribosomes, tRNAs and aminoacyl tRNA synthetases.

Reassigning more than a handful of sense codons across an entire genome remains challenging, as does incorporation of multiple non-standard amino acids into polypeptides with high purity.

After nine years of effort, the Church Lab demonstrated in 2013 that a codon could be freed in E. coli so the microbes could make a new amino acid; it took another 10 years to free a second codon, after which engineered cells could make proteins containing up to 22 different amino acids.

Synthetic genetic codes have enabled construction of organisms with new properties such as increased virus resistance and reduced horizontal gene transfer, as well as genetically encoded materials, therapeutics and biocatalysts.

The two freed codons took 19 years of Church Lab work between them, about nine and a half years per codon.

First author Felix Radford, HMS research fellow in genetics in the Church Lab, said AGENTEX lets researchers generate entirely new genetic codes on demand in test tubes and use them at scale, and that it is more rapid and safe than existing methods because it does not rely on handling living cells or altering their genomes.