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Synthetic biology strives to reliably control cellular behavior, typically in the form of user-designed interactions of biological components to produce a predetermined output. Engineered circuit components are frequently derived from natural sources and are therefore often hampered by inadvertent interactions with host machinery, most notably within the host central dogma. Reliable and predictable gene circuits require the targeted reduction or elimination of these undesirable interactions to mitigate negative consequences on host fitness and develop context-independent bioactivities. Here, we review recent advances in biological orthogonalization, namely the insulation of researcher-dictated bioactivities from host processes, with a focus on systematic developments that may culminate in the creation of an orthogonal central dogma and novel cellular functions. Development of fully synthetic nucleobase pairs that faithfully interact in living cells, and their applications in creating semisynthetic organisms with expanded and orthogonal information-carrying capacity.Harnessing naturally occurring and mutually orthogonal DNA replication systems to enable replication of target genes. Highly error-prone variations on these systems enable robust directed evolution of biomolecules.Engineering and directed evolution of mutually orthogonal transcription factors that operate with high dynamic range, low background, and respond to a wide repertoire of stimuli in vivo.Recent developments in in vivo orthogonal protein translation including: orthogonal RBS–orthogonal anti-RBS pairs, covalently linked rRNA subunits to discover novel enzymatic capabilities, improved incorporation of non-canonical amino acids and decoding quadruplet codons.

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo , and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. Random DNA mutagenesis provides genetic diversity both in nature and the laboratory. Here, Badran and Liu present a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that surpasses the mutational efficiency and spectra of the most widely used in vivo and in vitro mutagenesis methods.

The spontaneous deamination of cytosine is a major source of transitions from CG to TA base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted AT base pairs to GC could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of AT to GC in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPRCas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted AT base pairs efficiently to GC (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage. A new DNA ‘base editor’ can change targeted A•T base pairs to G•C, allowing disease-associated mutations to be corrected and disease-suppressing mutations to be introduced into cells. Base editing steps forward In 2016, David Liu and colleagues developed a DNA 'base editor'—a system that would make it possible to change C•G base pairs to T•A base pairs within DNA without introducing double-stranded breaks. This approach involves tethering of a cytidine deaminase to an inactive RNA-guided Cas9 complex that enables site selectivity. However, this system was unable to correct about half of the single nucleotide polymorphisms that are known to be pathogenic. Now, David Liu and collaborators describe the next step in genomic base editing technology, designed to tackle the conversion of A•T base pairs to G•C base pairs. Beginning with a bacterial adenosine deaminase that acts on RNA, they used seven rounds of selection and refinement to produce ABE7.10. This enzyme, again tethered to an inactive RNA-guided Cas9 complex, uses DNA as a substrate and resulted in an average correction efficiency of 53% across multiple sites and contexts in the genome, with a very low mutagenic background. Importantly, the system can be used both to correct disease-associated single nucleotide polymorphisms and to introduce disease-suppressing ones.

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

Antibiotic biosynthetic gene clusters (BGCs) produce bioactive metabolites that impart a fitness advantage to their producer, providing a mechanism for natural selection. This selection drives antibiotic evolution and adapts BGCs for expression in different organisms, potentially providing clues to improve heterologous expression of antibiotics. Here, we use phage-assisted continuous evolution (PACE) to achieve bioactivity-dependent adaptation of the BGC for the antibiotic bicyclomycin (BCM), facilitating improved production in a heterologous host. This proof-of-principle study demonstrates that features of natural bioactivity-dependent evolution can be engineered to access unforeseen routes of improving metabolic pathways and product yields. Biosynthetic gene clusters (BGCs) make small molecules with fitness-enhancing activities that drive BGC evolution. Here, the authors show that synthetic biology can leverage bioactivity to achieve continuous evolution of an antibiotic BGC in the lab and improve antibiotic production in a new host.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system. Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.

In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli , Pseudomonas aeruginosa , and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo. Ribosome kinetics are rate-limiting for protein synthesis. Here the authors evolve diverse 16S rRNAs for enhanced protein synthesis rates and genetic code expansion efficiencies in vivo.

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli . Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli -derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells. Synthetic biologists often co-opt heterologous parts to affect new functions in living cells, yet such an approach has rarely been extended to structural components of the ribosome. Here, the authors describe generalizable methods to express ribosomes from divergent microbes in E. coli and maximize their function.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein–protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant K d  = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. Phage-assisted continuous evolution (PACE) rapidly evolves Bacillus thuringiensis toxins through more than 500 generations of mutation, selection, and replication to bind a receptor expressed on the surface of insect-pest midgut cells. Beating Bt resistance in insect pests The emergence of insects resistant to Bacillus thuringiensis δ-endotoxins (Bt toxins) is threatening to reduce the effectiveness of this system in crops engineered to carry these insecticidal proteins. David Liu and colleagues have used phage-assisted continuous evolution (PACE) selection to rapidly evolve high-affinity protein–protein interactions, and applied the system to evolve Bt toxin variants that kill insects through binding a new insect gut cell protein target — a cadherin-like receptor from the insect pest Trichoplusia ni . The modified Bt toxins are shown to overcome Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.