Probing the Limits of Genetic Recoding in Essential Genes

Lajoie, Marc J.; Kosuri, Sriram; Mosberg, Joshua A.; Gregg, Christopher; Zhang, Di; Church, George M.

doi:10.1126/science.1241460

Cited by 125 publications

(162 citation statements)

References 37 publications

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“…Three of the N-terminal failures (ssb_AGA10, dnaT_AGA10, and prfB_AGG64) had RBS-like motifs that were either disrupted or created by CGU replacement. Although prfB_AGG64 is part of the RBS motif that triggers an essential frameshift mutation in prfB (21,38,39), pausing motifmediated regulation of ssb and dnaT expression has not been reported. Nevertheless, ribosomal pausing data (20) showed that ribosomal occupancy peaks are present directly downstream of the AGR codons for ssb and are absent for dnaT (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although Quax et al (18) provide an excellent review of how biology chooses codons, systematic and exhaustive studies of codon choice in whole genomes are lacking. Studies have only begun to probe the effects of codon choice empirically in a relatively small number of reporter genes (12,(19)(20)(21)(22). Several important questions must be answered as a first step toward designing custom genomes exhibiting new functions: How flexible is genomewide codon choice?…”

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confidence: 99%

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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: Resultsmentioning

confidence: 99%

mentioning

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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“…Whenever the drive is copied, the cut gene is replaced with a recoded version flanked by the other components of the drive. Recent work has demonstrated that most genes can be substantially recoded with little effect on organism fitness 93 ; the 3' untranslated region might be replaced with an equivalent sequence from a related gene. Because there would be no homology between the recoded cut site and the drive components, the drive cassette would always be copied as a unit.…”

Section: Evolutionary Stabilitymentioning

confidence: 99%

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

Esvelt

Smidler

Catteruccia

et al. 2014

Preprint

210

388

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Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

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“…Recoding is not permitted within the first 100 bp of each ORF, as these regions are known to have special preferences in terms of codon usage 7 . Together, these design rules favor high performance of almost all PCRTags under a single set of PCR conditions whereby synthetic and wild type PCRTag primer pairs exclusively bind and amplify synthetic and native DNA, respectively ( Figure 1B).…”

Section: Introductionmentioning

confidence: 99%