6 research outputs found
Defining synonymous codon compression schemes by genome recoding
Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome
Defining synonymous codon compression schemes by genome recoding
Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome
Total synthesis of Escherichia coli with a recoded genome
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
An artificial PPR scaffold for programmable RNA recognition
Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism in eukaryotic cells. Although recent computational and structural studies have provided insights into RNA recognition by PPR proteins, their highly insoluble nature and inconsistencies between predicted and observed modes of RNA binding have restricted our understanding of their biological functions and their use as tools. Here we use a consensus design strategy to create artificial PPR domains that are structurally robust and can be programmed for sequence-specific RNA binding. The atomic structures of these artificial PPR domains elucidate the structural basis for their stability and modelling of RNA-protein interactions provides mechanistic insights into the importance of RNA-binding residues and suggests modes of PPR-RNA association. The modular mode of RNA binding by PPR proteins holds great promise for the engineering of new tools to target RNA and to understand the mechanisms of gene regulation by natural PPR proteins
Heterozygosity for Roquinsan leads to angioimmunoblastic T-cell lymphoma-like tumors in mice
Angioimmunoblastic T-cell lymphoma
(AITL) is the second most common peripheral
T-cell lymphoma with unusual clinical
and pathologic features and a poor
prognosis despite intensive chemotherapy.
Recent studies have suggested
AITL derives from follicular helper T (TFH)
cells, but the causative molecular pathways
remain largely unknown. Here we
show that approximately 50% of mice
heterozygous for the “san” allele of
Roquin develop tumors accompanied by
hypergammaglobulinemia by 6 months of
age. Affected lymph nodes displayed the
histologic features diagnostic of AITL,
except for the presence of expanded FDC
networks. Accumulation of TFH cells preceded
tumor development, and clonal rearrangements
in the TCR-B genes were
present in most tumors. Furthermore, TFH
cells exhibited increased clonality compared
with non-TFH cells from the same
lymph nodes, even in the absence of
tumors. Genetic manipulations that prevent
TFH development, such as deletion of
ICOS, CD28, and SAP, partially or completely
abrogated tumor development,
confirming a TFH-derived origin. Roquinsan/+
mice emerge as a useful model to investigate
the molecular pathogenesis of AITL
and for preclinical testing of therapies
aimed at targeting dysregulated TFH cells
or their consequences.This work was supported by a Viertel Senior Medical Research
Fellowship and National Health and Medical Research Council
program and project grants (C.G.V.) and a National Health and
Medical Research Council Overseas Biomedical Fellowship (J.I.E.)