Kim C. Liu

Expanding the genetic code with diverse backbone structures across diverse sequence contexts

Carlos Piedrafita, A. D. Dickson, Daniel U. Richter, Caroline Weber et autres

Abstract Expanding the genetic code to enable the selective and specific incorporation of non-canonical monomers (ncMs), beyond α-L amino acids with variant sidechains, is a key outstanding challenge. Here we discover orthogonal aminoacyl-tRNA synthetases that selectively and specifically acylate their cognate orthogonal …

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A portable orthogonal replication system enables continuous gene evolution near the biological speed limit

Rongzhen Tian, Fabian B. H. Rehm, Matthew Kenneth, Kiarash Jamali et autres

Abstract Orthogonal DNA replication systems uncouple the mutagenesis of target genes from host viability, enabling target gene hypermutation beyond the genomic critical error threshold and thereby unlocking access to greater sequence space for accelerated evolution. Here we introduce a series of upgrades …

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Highly mutagenic continuous evolution in E. coli using a Φ29-based orthogonal replication system

Fabian B. H. Rehm, Kim C. Liu, Rongzhen Tian, Jason W. Chin

Abstract Organisms that permit hypermutation of target genes without off-target mutagenesis of the host genome enable the accelerated, continuous evolution of genes for new or enhanced functions. We develop and optimize an orthogonal DNA replication system in Escherichia coli that uses components …

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DNA supercoiling links transcription and chromatin architecture during human stem cell differentiation

Consuelo Perez, Pierre Murat, Andrew Zeller, Kim C. Liu et autres

Abstract Transcription imposes torsional stress on chromatin, leading to over– or under-winding of the DNA helix. Yet, how supercoiling evolves during dynamic changes in gene expression, and how it influences three-dimensional chromatin contacts in the densely packed human genome, remains unclear. Here, …

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High-fidelity human chromosome transfer and elimination

Gianluca Petris, Simona Grazioli, Linda van Bijsterveldt, Pierre Murat et autres

The synthesis of human genomes and other gigabase-scale genomes will require new strategies. Here, we realized key steps in our pipeline for building synthetic human chromosomes. We established: (i) the facile transfer of human chromosomes from human cells to mouse embryonic stem …

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Escherichia coli with a 57-codon genetic code

Wesley E. Robertson, Fabian B. H. Rehm, Martin Spinck, R. R. Schumann et autres

The near-universal genetic code uses 64 codons to encode the 20 canonical amino acids and protein synthesis. Here, we designed and generated Escherichia coli with a 4-megabase synthetic genome in which we replaced known occurrences of six sense codons and a stop …

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Genetic Code-Locking Confers Stable Virus Resistance to a Recoded Organism

Jérôme F. Zürcher, A. D. Dickson, Tomás Kappes, Askar A. Kleefeldt et autres

The genetic code defines the correspondence between codons in genes and amino acids in proteins. Reassignment of sense codons to different amino acids can create cells with refactored genetic codes that are distinct from the canonical genetic code. By encoding essential genes …

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Escherichia coli with a 57-codon genetic code

Wesley E. Robertson, Fabian B. H. Rehm, Martin Spinck, R. R. Schumann et autres

Abstract The near-universal genetic code of living organisms uses 64 codons to encode the 20 canonical amino acids in protein synthesis. Here we design and generate a variant of Escherichia coli with a 4 Mb synthetic genome in which we replace every …

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Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli

Rongzhen Tian, Fabian B. H. Rehm, Dariusz Czernecki, Yangqi Gu et autres

The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and …

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Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Daniel L. Dunkelmann, Carlos Piedrafita, A. D. Dickson, Kim C. Liu et autres

Abstract The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1–3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases …

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Triplet-Encoded Prebiotic RNA Aminoacylation

Meng Su, Christian Schmitt, Ziwei Liu, Samuel J. Roberts et autres

The encoding step of translation involves attachment of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases, themselves the product of coded peptide synthesis. So, the question arises─before these enzymes evolved, how were primordial tRNAs selectively aminoacylated? Here, we demonstrate enzyme-free, sequence-dependent, chemoselective …

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Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly

Jérôme F. Zürcher, Askar A. Kleefeldt, Louise F. H. Funke, Jakob Birnbaum et autres

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