Overcoming Challenges in Engineering the Genetic Code

Lajoie, Marc J.; Söll, Dieter; Church, George M.

doi:10.1016/j.jmb.2015.09.003

Cited by 50 publications

(53 citation statements)

References 194 publications

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“…4) suggests that our genome design guidelines provide a strong first approximation of acceptable modifications to the primary sequence of viable genomes. These design rules coupled with inexpensive DNA synthesis will facilitate the construction of radically redesigned genomes exhibiting useful properties such as biocontainment, virus resistance, and expanded amino acid repertoires (45).…”

Section: Discussionmentioning

confidence: 99%

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.codon choice | genome editing | recoded genomes

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Section: Discussionmentioning

confidence: 99%

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Napolitano

Landon

Gregg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The stage is set for developing a broader array of OTSs for physiologically relevant PTMs with an eye towards multi-site incorporation of different PTMs. The future of PTM chemical biology in this arena will require more recoding to reassign more codons as well as engineering new sets of OTS elements that can decipher various codons that are not cross-reactive 12,13 .…”

Section: Except After Cmentioning

confidence: 99%

“…Progress has been made toward more extensively recoded organisms with multiple codons available for ncAA incorporation (Fig. 3a), and the development of organisms utilizing genomic nonstandard base pairs or orthogonal ribosomes that decode quadruplet codons provide entirely new codons to encode ncAAs 7,13,20 although these organisms raise questions of how many ncAAs incorporated in the same protein will prove interesting or relevant. Still, differences in protein folding and compartmentalization between human cells and other organisms will continue to impede the production of many functional recombinant human proteins.…”

Section: As In Neighbor and Weighmentioning

confidence: 99%

The ABCs of PTMs

Barber

Rinehart

2018

Nat Chem Biol

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“…The number of developed OTSs has rapidly expanded to include a large number of orthogonal aaRS•tRNA pairs [1-3]. While most GCE studies have focused on the suppression of the three stop codons (UAA, UAG, UGA), an increasing number of studies have targeted sense codons for reassignment [4, 5]. …”

Section: Introductionmentioning

confidence: 99%

The central role of tRNA in genetic code expansion

Reynolds

Vargas-Rodriguez

Söll

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation. Scope of Review Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins. Major Conclusions The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio. General Significance Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions.

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Overcoming Challenges in Engineering the Genetic Code

Cited by 50 publications

References 194 publications

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

The ABCs of PTMs

The central role of tRNA in genetic code expansion

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