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Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications 1 – 3 . However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene 4 – 6 . Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics. The feasibility of adeno-associated-virus-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) is explored to restore gene function, using a mouse model of mucopolysaccharidosis type I for proof of concept.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA. High-fidelity convergent total synthesis is used to produce Escherichia coli with a 61-codon synthetic genome that uses 59 codons to encode all of the canonical amino acids.

All eukaryotes utilize a single termination factor, eRF1, to halt translation when the ribosome encounters one of three possible stop codons; here electron cryo-microscopy structures of ribosome–eRF1 complexes in the process of recognizing each stop codon reveal how stop codons are discriminated from sense codons. How mRNA knows when to stop Mammalian messenger RNAs utilize three stop codons, but have a single termination factor, eRF1, that can recognize all three. To understand how eRF1 can distinguish stop codons from sense codons, Alan Brown et al . determined the structures of the mammalian 80S ribosome bound to eRF1 and mRNAs containing each of the stop codons. They find that two nucleotides from the 18S rRNA are stacked with two of the stop codon nucleotides, and the next nucleotide, to compact the mRNA, a conformation that favours stop codons to the exclusion of sense codons. Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases 1 . By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons 2 . The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5–3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination 3 , 4 .

We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Most amino acids are encoded by multiple codons, and codon choice has strong effects on protein expression. Rare codons are enriched at the N terminus of genes in most organisms, although the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in Escherichia coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by “endoreduplication intercross.” Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle 1 – 4 . Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop . We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAs Glu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNA Trp CCA from five to four base pairs (bp). The canonical 5-bp tRNA Trp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop , Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum . Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons. Analyses of in-frame stop codons in protein-coding genes of Blastocrithidia nonstop with all three stop codons reassigned reveal a mechanism for UGA reassignment in eukaryotes involving shortening of the tRNA anticodon stem and a mutant eRF1 release factor.

Early STOP codons created with CRISPR base editors leads to gene knockout with high efficiency and does not stress cells with double-strand DNA breaks. CRISPR-STOP can target the majority of human genes and is useful for genetic screens. CRISPR–Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors 1 , 2 – 3 . Inspired by this natural malleability, synthetic approaches have demonstrated whole-genome replacement of synonymous codons to construct genomically recoded organisms (GROs) 4 , 5 with alternative genetic codes. However, no efforts have fully leveraged translation factor plasticity and codon degeneracy to compress translation function to a single codon and assess the possibility of a non-degenerate code. Here we describe construction and characterization of Ochre, a GRO that fully compresses a translational function into a single codon. We replaced 1,195 TGA stop codons with the synonymous TAA in ∆TAG Escherichia coli C321.∆A 4 . We then engineered release factor 2 (RF2) and tRNA Trp to mitigate native UGA recognition, translationally isolating four codons for non-degenerate functions. Ochre thus utilizes UAA as the sole stop codon, with UGG encoding tryptophan and UAG and UGA reassigned for multi-site incorporation of two distinct non-standard amino acids into single proteins with more than 99% accuracy. Ochre fully compresses degenerate stop codons into a single codon and represents an important step toward a 64-codon non-degenerate code that will enable precise production of multi-functional synthetic proteins with unnatural encoded chemistries and broad utility in biotechnology and biotherapeutics. Ochre, a strain of Escherichia coli engineered to have a single stop codon, enables reassignment of four codons for non-degenerate functions, such as incorporation of non-standard amino acids into proteins.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidinoquinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).