RESUMO
Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.
Assuntos
Aldose-Cetose Isomerases/química , Evolução Molecular Direcionada/métodos , Resistência a Medicamentos/genética , Malária/tratamento farmacológico , Mutagênese , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/metabolismo , Antibacterianos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacologia , Biblioteca Gênica , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The ErbB family of tyrosine kinase receptors is a key element in preserving cell growth homeostasis. This family is comprised of four single-transmembrane domain proteins designated ErbB-1-4. Ligand binding initiates dimerization followed by tyrosine phosphorylation and signaling, which when uncontrolled lead to cancer. Accordingly, extensive research has been devoted to finding ErbB-intercepting agents, directed against ErbB-1 and ErbB-2, but so far, no inhibitor has targeted the transmembrane domain (TMD), which is instrumental for receptor dimerization and activation. Moreover, no antitumor agents targeted ErbB-3, which although it cannot generate signals in isolation, its heterodimerization with ErbB-2 leads to the most powerful and oncogenic signaling unit in the ErbB family. Here, to further elucidate the role of the interactions between the TMDs of the ErbB family in cancer, we investigated peptides derived from the TMDs of ErbB-1 and ErbB-2. We then focused on the C-terminal domains (B2C) and their analogue, named B2C-D, that contains both d- and l-amino acids. Both peptides incorporated the distal GXXXG dimerization motif to target the TMD of ErbB-3. Our results revealed that B2C-D is highly active both in vitro and in vivo. It significantly inhibits neuregulin- and EGF-induced ErbB activation, impedes the proliferation of a battery of human cancer cell lines, and retards tumor growth in vivo. Notably, combining low concentrations of B2C-D and gemcitabine chemotherapy completely arrested proliferation of pancreatic cancer cells. Biochemical and in vitro interaction studies suggest direct interference with the assembly of the wild-type ErbB-2-ErbB-3 heterodimer as the underlying mode of action. To the best of our knowledge, this is the first agent to target the TMDs of ErbB to delay tumor growth and signaling.
Assuntos
Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/patologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dimerização , Humanos , Técnicas In Vitro , Proteínas de Membrana/química , Neoplasias/metabolismo , Peptídeos/química , Fosforilação , Homologia de Sequência de AminoácidosRESUMO
The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glândulas Mamárias Humanas/citologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Análise em Microsséries , Proteoma/análise , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Bactrocera zonata, a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a significant threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent it spreading, monitoring efforts in cultivation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional laboratories. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata. RESULTS: We demonstrate the specificity and high sensitivity of this method. Identification of target pests was done using specific and universal primers on pooled samples, enabling differentiation of pests with high certainty. We also demonstrate reaction stability over time for future on-site applications. DISCUSSION: Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification and Cas12a-based detection. This method is highly modular, and the presented target design method can be applied to a wide array of pests. This approach can be easily adapted to fit local threats and requires minimal training of operators in border controls and other relevant locations, reshaping pest control and making state-of-the-art technologies available worldwide, including in developing countries. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Sistemas CRISPR-Cas , Núcleo Familiar , Europa (Continente) , Ásia Oriental , AustráliaRESUMO
Monitoring astronauts' health during space missions poses many challenges, including rapid assessment of crew health conditions. Sensitive genetic diagnostics are crucial for examining crew members and the spacecraft environment. CRISPR-Cas12a, coupled with isothermal amplification, has proven to be a promising biosensing system for rapid, on-site detection of genomic targets. However, the efficiency and sensitivity of CRISPR-based diagnostics have never been tested in microgravity. We tested the use of recombinase polymerase amplification (RPA) coupled with the collateral cleavage activity of Cas12a for genetic diagnostics onboard the International Space Station. We explored the detection sensitivity of amplified and unamplified target DNA. By coupling RPA with Cas12a, we identified targets in attomolar concentrations. We further assessed the reactions' stability following long-term storage. Our results demonstrate that CRISPR-based detection is a powerful tool for on-site genetic diagnostics in microgravity, and can be further utilized for long-term space endeavors to improve astronauts' health and well-being.
Assuntos
Técnicas Biossensoriais , Ausência de Peso , Humanos , Astronautas , Genômica , Recombinases , Sistemas CRISPR-Cas/genética , Técnicas de Amplificação de Ácido NucleicoRESUMO
Saturation mutagenesis is a semi-rational approach for protein engineering where sites are saturated either entirely or partially to include amino acids of interest. We previously reported on a codon compression algorithm, where a set of minimal degenerate codons are selected according to user-defined parameters such as the target organism, type of saturation and usage levels. Here, we communicate an addition to our web tool that considers the distance between the wild-type codon and the library, depending on its purpose. These forms of restricted collections further reduce library size, lowering downstream screening efforts or, in turn, allowing more comprehensive saturation of multiple sites. The library design tool can be accessed via http://www.dynamcc.com/dynamcc_d/. Graphical Abstract.
RESUMO
Functional genomics is an essential tool for elucidating the structure and function of genes in any living organism. Here, we review the use of different gene manipulation techniques in functional genomics of Acari (mites and ticks). Some of these Acari species inflict severe economic losses to managed crops and health problems to humans, wild and domestic animals, but many also provide important ecosystem services worldwide. Currently, RNA interference (RNAi) is the leading gene expression manipulation tool followed by gene editing via the bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 system (CRISPR-Cas9). Whilst RNAi, via siRNA, does not always lead to expected outcomes, the exploitations of the CRISPR systems in Acari are still in their infancy and are limited only to CRISP/Cas9 to date. In this review, we discuss the advantages and disadvantages of RNAi and CRISPR-Cas9 and the technical challenges associated with their exploitations. We also compare the biochemical machinery of RNAi and CRISPR-Cas9 technologies. We highlight some potential solutions for experimental optimization of each mechanism in gene function studies. The potential benefits of adopting various CRISPR-Cas9 systems for expanding on functional genomics experiments in Acari are also discussed.
Assuntos
Ácaros e Carrapatos , Sistemas CRISPR-Cas , Animais , Ecossistema , Edição de Genes/métodos , Genômica/métodosRESUMO
Ligand-induced dimerization of the epidermal growth factor receptor (ErbB-1/EGFR) involves conformational changes that expose an extracellular dimerization interface. Subsequent alterations within the cytoplasmic kinase domain, which culminate in tyrosine phosphorylation, are less understood. Our study addressed this question by using two strategies: a chimeric receptor approach employed ErbB-3, whose defective kinase domain was replaced by the respective part of EGFR. The implanted full-length kinase, unlike its subdomains, conferred dimerization and catalysis. The data infer that the kinase function of EGFR is restrained by the carboxyl tail; once grafted distally to the ectopic tail of ErbB-3, the kinase domain acquires quasi-dimerization and activation. In an attempt to alternatively refold the cytoplasmic tail, our other approach employed kinase inhibitors. Biophysical measurements and covalent cross-linking analyses showed that inhibitors targeting the active conformation of EGFR, in contrast to a compound recognizing the inactive conformation, induce quasi-dimers in a manner similar to the chimeric ErbB-3 molecule. Collectively, these observations unveil kinase domain-mediated quasi-dimers, which are regulated by an autoinhibitory carboxyl tail. On the basis of these observations, we propose that quasi-dimers precede formation of ligand-induced, fully active dimers, which are stabilized by both extracellular and intracellular receptor-receptor interactions.
Assuntos
Receptores ErbB/metabolismo , Fosfotransferases/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/genética , Transferência Ressonante de Energia de Fluorescência , Mutagênese Sítio-Dirigida , Fosfotransferases/genética , Multimerização Proteica/genética , Estrutura Terciária de Proteína , Receptor ErbB-3/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.
RESUMO
Saturation mutagenesis is conveniently located between the two extremes of protein engineering, namely random mutagenesis, and rational design. It involves mutating a confined number of target residues to other amino acids, and hence requires knowledge regarding the sites for mutagenesis, but not their final identity. There are many different strategies for performing and designing such experiments, ranging from simple single degenerate codons to codon collections that code for distinct sets of amino acids. Here, we provide detailed information on the Dynamic Management for Codon Compression (DYNAMCC) approaches that allow us to precisely define the desired amino acid composition to be introduced to a specific target site. DYNAMCC allows us to set usage thresholds and to eliminate undesirable stop and wild-type codons, thus allowing us to control library size and subsequently downstream screening efforts. The DYNAMCC algorithms are free of charge and are implemented in a website for easy access and usage: www.dynamcc.com .
Assuntos
Códon/genética , Mutagênese/genética , Algoritmos , Aminoácidos/genética , Compressão de Dados/métodos , Biblioteca Gênica , Engenharia de Proteínas/métodosRESUMO
Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.
Assuntos
Aldose-Cetose Isomerases/genética , Antimaláricos/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Fosfomicina/análogos & derivados , Aldose-Cetose Isomerases/metabolismo , Antimaláricos/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/química , Escherichia coli/metabolismo , Fosfomicina/metabolismo , Fosfomicina/farmacologia , Engenharia Genética/métodos , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Plasmodium falciparum/efeitos dos fármacosRESUMO
Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.
Assuntos
Citoesqueleto de Actina/metabolismo , Autoantígenos/metabolismo , Neoplasias da Mama/patologia , Mama/patologia , Fator de Crescimento Epidérmico/farmacologia , Filaminas/metabolismo , Colágenos não Fibrilares/metabolismo , Proteômica/métodos , Animais , Autoantígenos/genética , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Receptores ErbB/metabolismo , Feminino , Filaminas/genética , Humanos , Marcação por Isótopo , Camundongos , Camundongos Nus , Colágenos não Fibrilares/genética , Fosforilação , Ligação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto , Colágeno Tipo XVIIRESUMO
Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.
Assuntos
Algoritmos , Engenharia Genética , Escherichia coli/genética , Biblioteca Gênica , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Plasmídeos/genética , Plasmídeos/metabolismo , Análise de Sequência de DNARESUMO
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology.IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.
Assuntos
Evolução Molecular Direcionada , Código Genético , Mutação Puntual , Biologia Sintética/métodos , Modelos GenéticosRESUMO
Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.
Assuntos
Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Edição de Genes/métodos , Engenharia Metabólica/métodos , Polimorfismo de Nucleotídeo Único/genética , Engenharia de Proteínas/métodos , Algoritmos , Genoma Bacteriano/genética , Genoma Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Metaboloma/genética , Nucleotídeos/genética , Proteoma/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SoftwareRESUMO
The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of information. To address this issue, we introduce the Resistome database: a literature-curated collection of Escherichia coli genotypes-phenotypes containing over 5,000 mutants that resist hundreds of compounds and environmental conditions. We use the Resistome to understand our current state of knowledge regarding resistance and to detect potential synergy or antagonism between resistance phenotypes. Our data set represents one of the most comprehensive collections of genomic data related to resistance currently available. Future development will focus on the construction of a combined genomic-transcriptomic-proteomic framework for understanding E. coli's resistance biology. The Resistome can be downloaded at https://bitbucket.org/jdwinkler/resistome_release/overview .
Assuntos
Bases de Dados Factuais , Escherichia coli , Adaptação Biológica/genética , Adaptação Biológica/fisiologia , Farmacorresistência Bacteriana , Epistasia Genética , Escherichia coli/genética , Escherichia coli/fisiologia , Genótipo , Aprendizado de Máquina , Mutação , Pressão Osmótica , FenótipoRESUMO
Saturation mutagenesis is widely used in protein engineering and other experiments. A common practice is to utilize the single degenerate codon NNK. However, this approach suffers from amino acid bias and the presence of a stop codon and of the wild type amino acid. These extra features needlessly increase library size and consequently downstream screening load. Recently, we developed the DYNAMCC algorithms for codon compression that find the minimal set of degenerate codons, covering any defined set of amino acids, with no off-target codons and with redundancy control. Additionally, we experimentally demonstrated the advantages of this approach over the standard NNK method. While the code is freely available from our Web site, we have now made this method more accessible to a broader audience without any computational background by building a user-friendly web-based interface for those algorithms. The Web site can be accessed through: www.dynamcc.com .
Assuntos
Códon/genética , Código Genético/genética , Algoritmos , Aminoácidos/genética , Biblioteca Gênica , Mutagênese/genética , Engenharia de Proteínas/métodosRESUMO
The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.
Assuntos
Bactérias/genética , Bacteriófago lambda/genética , Engenharia Genética , Recombinação Homóloga , Biologia SintéticaRESUMO
Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency.
Assuntos
Códon/genética , Mutagênese/genética , Algoritmos , Aminoácidos/genética , Biblioteca Gênica , Mutação/genética , Oligonucleotídeos/genética , Engenharia de Proteínas/métodosRESUMO
Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.