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Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties. This review categorizes current continuous evolution into three distinct classes: non-targeted chromosomal, targeted chromosomal, and extra-chromosomal hypermutation approaches. It also compares various continuous evolution strategies based on different principles, providing a reference for selecting suitable methods for specific evolutionary goals. Furthermore, this review discusses the two primary limitations for further widespread application of in vivo continuous evolution, which are lack of general applicability and insufficient mutagenic capability. We envision that developing generally applicable mutagenic components and methods to enhance mutation rates for in vivo continuous evolution are promising future directions for wide range applications of continuous evolution.

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Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions.

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Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.

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Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.

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Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

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Phage therapy or Phage treatment is the use of bacteriolysing phage in treating bacterial infections by using the viruses that infects and kills bacteria. This technique has been studied and practiced very long ago, but with the advent of antibiotics, it has been neglected. This foregone technique is now witnessing a revival due to development of bacterial resistance. Nowadays, with the awareness of genetic sequence of organisms, it is required that informed choices of phages have to be made for the most efficacious results. Furthermore, phages with the evolving genes are taken into consideration for the subsequent improvement in treating the patients for bacterial diseases. In addition, direct evolution methods are increasingly developing, since these are capable of creating new biological molecules having changed or unique activities, such as, improved target specificity, evolution of novel proteins with new catalytic properties or creation of nucleic acids that are capable of recognizing required pathogenic bacteria. This system is incorporates continuous evolution such as protein or genes are put under continuous evolution by providing continuous mutagenesis with least human intervention. Although, this system providing continuous directed evolution is very effective, it imposes some challenges due to requirement of heavy investment of time and resources. This chapter focuses on development of phage as a therapeutic agent against various bacteria causing diseases and it improvement using direct evolution of proteins and nucleic acids such that they target specific organisms.

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Directed evolution is a powerful technique that uses principles of natural evolution to enable the development of biomolecules with novel functions. However, the slow pace of natural evolution does not support the demand for rapidly generating new biomolecular functions in the laboratory. Viruses offer a unique path to design fast laboratory evolution experiments, owing to their innate ability to evolve much more rapidly than most living organisms, facilitated by a smaller genome size that tolerate a high frequency of mutations, as well as a fast rate of replication. These attributes offer a great opportunity to evolve various biomolecules by linking their activity to the replication of a suitable virus. This review highlights the recent advances in the application of virus-assisted directed evolution of designer biomolecules in both prokaryotic and eukaryotic cells.

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Directed evolution has become one of the most successful and powerful tools for protein engineering. However, the efforts required for designing, constructing, and screening a large library of variants can be laborious, time-consuming, and costly. With the recent advent of machine learning (ML) in the directed evolution of proteins, researchers can now evaluate variants in silico and guide a more efficient directed evolution campaign. Furthermore, recent advancements in laboratory automation have enabled the rapid execution of long, complex experiments for high-throughput data acquisition in both industrial and academic settings, thus providing the means to collect a large quantity of data required to develop ML models for protein engineering. In this perspective, we propose a closed-loop in vitro continuous protein evolution framework that leverages the best of both worlds, ML and automation, and provide a brief overview of the recent developments in the field.

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Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

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Noncovalent interactions between biomolecules such as proteins and nucleic acids coordinate all cellular processes through changes in proximity. Tools that perturb these interactions are and will continue to be highly valuable for basic and translational scientific endeavors. By taking cues from natural systems, such as the adaptive immune system, we can design directed evolution platforms that can generate proteins that bind to biomolecules of interest. In recent years, the platforms used to direct the evolution of biomolecular binders have greatly expanded the range of types of interactions one can evolve. Herein, we review recent advances in methods to evolve protein-protein, protein-RNA, and protein-DNA interactions.

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Selecting desired variants from protein libraries is always a challenge for directed evolution. Engineering synthetic auxotrophs to establish a link between cell growth and protein property allows growth-coupled in vivo selection, which is high throughput and compatible with continuous evolution. In silico simulation-guided metabolic reprogramming will help in the design of customized synthetic auxotrophs.

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Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

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Traditional approaches to the directed evolution of genes of interest (GOIs) place constraints on the scale of experimentation and depth of evolutionary search reasonably achieved. Engineered genetic systems that dramatically elevate the mutation of target GOIs in vivo relieve these constraints by enabling continuous evolution, affording new strategies in the exploration of sequence space and fitness landscapes for GOIs. We describe various in vivo hypermutation systems for continuous evolution, discuss how different architectures for in vivo hypermutation facilitate evolutionary search scale and depth in their application to problems in protein evolution and engineering, and outline future opportunities for the field.

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Protein-protein interactions (PPIs) are involved in nearly all cellular processes. PPIs are particularly crucial for mediating selectivity along signaling pathways. Thus, measuring the competitive interplay between PPIs in a cell is important for both understanding fundamental cellular regulation and developing therapeutics targeting those whose dysregulation is associated with disease. A variety of split protein reporter-based tools are available to measure if two proteins interact within a cell and thereby characterize the general determinants of their interactions. PPIs, however, occur within complex networks facilitated by dynamic biophysical nuances that determine activity and selectivity. Evolved, proximity-dependent split T7 RNA polymerase (RNAP) biosensors have recently been used to perform deep mutational scanning of PPI interfaces, and to create synthetic gene circuits. In this chapter, we present the application of proximity-dependent split RNAP biosensors as a method to measure multidimensional PPIs in live cells. Orthogonal split RNAP "tags" encode each interaction in a unique RNA signal, thereby enabling the study of multiple competitive PPIs in live cells. Each unique RNA signal can be quantified via established RNA analysis methods. Herein, we provide advice and protocols to aid other researchers in using the split RNAP biosensor, focusing primarily on how to detect multiple PPIs in mammalian cells, including their dynamic interplay in the presence of small molecule inhibitors.

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We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multiparameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multimutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

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Evolutionary approaches have been providing solutions to various bioengineering challenges in an efficient manner. In addition to traditional adaptive laboratory evolution and directed evolution, recent advances in synthetic biology and fluidic systems have opened a new era of evolutionary engineering. Synthetic genetic circuits have been created to control mutagenesis and enable screening of various phenotypes, particularly metabolite production. Fluidic systems can be used for high-throughput screening and multiplexed continuous cultivation of microorganisms. Moreover, continuous directed evolution has been achieved by combining all the steps of evolutionary engineering. Overall, modern tools and systems for evolutionary engineering can be used to establish the artificial equivalent to natural evolution for various research applications.

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We recently developed an orthogonal replication system (OrthoRep) in yeast that allows for the rapid continuous mutagenesis of a special plasmid without mutating the genome. Although OrthoRep has been successfully applied to evolve several proteins and enzymes, the generality of OrthoRep has not yet been systematically studied. Here, we show that OrthoRep is fully compatible with all Saccharomyces cerevisiae strains tested, demonstrate that the orthogonal plasmid can encode genetic material of at least 22 kb, and report a CRISPR/Cas9-based method for expedient genetic manipulations of OrthoRep. It was previously reported that the replication system upon which OrthoRep is based is only stable in respiration-deficient S. cerevisiae strains that have lost their mitochondrial genome (ρ0 strains). However, here we trace this biological incompatibility to the activity of the dispensable toxin/antitoxin system encoded on the wild-type orthogonal plasmid. Since the toxin/antitoxin system is replaced by genes of interest in any OrthoRep application, OrthoRep is a generally compatible platform for continuous in vivo evolution in S. cerevisiae.

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Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.

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Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.

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Genetic diversity promotes laboratory based evolution and researchers can use mutagenesis and artificial selection to acquire expected phenotypes more frequently. Continuous evolution makes the important steps of laboratory evolution integrated into an uninterrupted cycle, enabling more generations to be evolved and scarcely any human interventions. Phages such as X174, T7 and λ bacteriophages with ideal mutation rates and feasibility to manipulate have been developed as an excellent platform for in vivo continuous evolution. Researchers at Harvard have recently invented an innovative system for the directed evolution of interested biomolecules: phage-assisted continuous evolution (PACE). PACE enables researchers to evolve biomolecules with desired properties within a very short time, which can perform more than 40 rounds of evolution in one day. Up to now, RNAP, protease, gene editing tool, receptor binding protein, aminoacyl-tRNA synthetase and single-chain variable fragments (scFv) have been optimized and evolved with improved activities and specificities by PACE. In addition, two simplified versions of PACE (PRECEL, PANCE) and a novel M13 phagemid-based system have been developed to expand the toolkit of directed evolution. It is promising and exciting that potentially any disease-related molecules which link desired activities with phage propagation can be optimized by above phage-assisted systems, thus leading to more potent biological therapies to be developed and applied into the clinic.