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At least 19 recordsLinked to original sources
PubMed2025

Bacterial directed evolution of CRISPR base editors.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.

Gene Editing↗
PubMed2017

A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line.

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

Capsid↗
PubMed2026

Directed evolution of Lactiplantibacillus plantarum for utilizing ethanol to produce postbiotics.

Alcohol is a recognized carcinogen worldwide. In this study, we aimed to utilize probiotics to metabolize ethanol and produce postbiotics. Initially, we identified a lactic acid bacteria community in kimchi with excellent probiotic activity. By employing our previously developed directed evolution techniques, a Lactiplantibacillus plantarum mutant with safe characteristics and an ethanol utilization capacity of 40 g/L and 0.15 g/L/OD was obtained. Genome sequencing and RT-qPCR analysis revealed the up-regulated expression of alcohol dehydrogenase and aldehyde dehydrogenase genes greatly contributed to ethanol utilization. Furthermore, the mutant strain demonstrated marked superiority in producing postbiotics, including antimicrobial peptides and beneficial organic acids such as lactic acid, phenyllactic acid, succinic acid, and indole-3-lactic acid. In the ethanol-fed fermentation process, the mutant strain achieved a lactic acid yield of 8.47 g/L and a carbon conversion rate of 21.8%. In vivo testing further validated its safety and ability to assist alcohol metabolism.

Adaptive laboratory evolution↗
PubMed1978

Regulation of newly evolved enzymes. IV. Directed evolution of the Ebg repressor.

In Escherichia coli, the wild-type repressor of ebg (evolved beta-galactosidase) enzyme synthesis, specified by the ebgR+ gene, responds very weakly to lactulose (fructose-beta-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 X 10(-10) . EBGR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

Biological Evolution↗
PubMed1972

Directed evolution of metabolic pathways in microbial populations. I. Modification of the acid phosphatase pH optimum in S. cerevisiae.

An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.-The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 10(9) cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (K(m)) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.-The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.-The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained.

Acid Phosphatase↗
PubMed2024

Directed evolution of engineered virus-like particles with improved production and transduction efficiencies.

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of proteins and RNAs, including gene editing agents. We report a system for the laboratory evolution of eVLPs that enables the discovery of eVLP variants with improved properties. The system uses barcoded guide RNAs loaded within DNA-free eVLP-packaged cargos to uniquely label each eVLP variant in a library, enabling the identification of desired variants following selections for desired properties. We applied this system to mutate and select eVLP capsids with improved eVLP production properties or transduction efficiencies in human cells. By combining beneficial capsid mutations, we developed fifth-generation (v5) eVLPs, which exhibit a 2-4-fold increase in cultured mammalian cell delivery potency compared to previous-best v4 eVLPs. Analyses of v5 eVLPs suggest that these capsid mutations optimize packaging and delivery of desired ribonucleoprotein cargos rather than native viral genomes and substantially alter eVLP capsid structure. These findings suggest the potential of barcoded eVLP evolution to support the development of improved eVLPs.

Humans↗
PubMed1992

Improvement by benzoquinones of the quantum yield of photoactivation of photosynthetic oxygen evolution: direct evidence for the two-quantum mechanism.

Effects of eight differently substituted 1,4-benzoquinones (BQs) on the quantum yield of photoactivation of oxygen evolution (reconstitution of the Mn cluster) were examined with wheat photosystem II (PSII) membranes depleted of the Mn cluster by treatment with 1.0 mM NH2OH. Illumination with 10 flashes at 0.25-s intervals of the PSII membranes in the presence of 2.0 mM Mn2+, 20 mM Ca2+, and 1.2 M Cl- restored 14% of oxygen-evolving activity destroyed by the NH2OH treatment. Among the benzoquinones tested, DBMIB (2,5-dibromo-3-methyl-6-isopropyl-BQ) and tetramethyl-BQ did not enhance the activity recovery, but all the others doubled the recovery when present at their optimal concentrations during illumination. The order of effectiveness was tetrabromo-, phenyl-, and 2,6-dichloro-BQs greater than or equal to 2,5-dichloro-BQ greater than tetrachloro-BQ greater than 2,5-dimethyl-BQ, though the differences were small. This order reflects their efficiencies as electron acceptors of PSII. This finding, together with others, suggests that the enhancement of activity recovery results from rapid oxidation by the benzoquinones of the reduced form of the quinone acceptors in PSII, QA- and QB-, which cause loss of an oxidized intermediate through charge-recombination reaction with Mn3+. The flash-number dependence of the recovery of oxygen-evolving activity indicated that the activity was not restored after one flash but recovered significantly after illumination with two flashes and then further increased upon additional flashes. This provides direct evidence that the minimum quantum requirement for photoactivation is two.

Benzoquinones↗
PubMed1989

An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.

Alkaline Phosphatase↗
PubMed1992

Directed evolution of an RNA enzyme.

An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

Animals↗
PubMed2009

Directed evolution of adeno-associated virus for glioma cell transduction.

Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.

Capsid Proteins↗
PubMed1986

Rapid electrocatalytic procedure for hydrogenase kinetic determination in the H2 evolution direction.

The linear sweep voltammetric method is used as a new approach for kinetic determination with enzymes accepting reversible redox couples as cosubstrate. A monolayer of hydrogenase molecules is grafted onto a glassy carbon electrode which is both the support of the enzyme and the detector of the activity. Reduced viologen concentration in the enzyme microenvironment is controlled by the electrode potential. The catalytic current produced by the enzyme allows an easy kinetic constant determination without the classical constraints found in hydrogenase kinetic measurements.

Catalysis↗
PubMed1992

Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.

Amino Acid Sequence↗
PubMed2022

Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution.

PURPOSE: Retinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge. METHODS: We introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes. Using the Tg(Cspg4-DsRed.T1)1Akik/J reporter mouse model, which has red fluorescent pericytes that can be isolated via flow cytometry in order to recover vector genomes, we performed three rounds of screening and identified seven putative mutants capable of transducing retinal pericytes. RESULTS: Following intravitreal administration of mutant vectors packaging ubiquitously expressing green fluorescent protein reporters and postmortem flow cytometry of enzymatically digested retinae, two mutants in particular, Peri-E and Peri-G, demonstrated significantly greater transduction of retinal pericytes than unmodified rAAV2/2 (1.4-fold and 2.8-fold, respectively). CONCLUSIONS: Although difficult to characterize the effect of each point mutation in the context of multiple amino acid variations from the wild-type AAV2 sequence, we identified several point mutations that may play critical roles in limiting HSPG binding, evading neutralization by murine A20 monoclonal antibodies, modulating antigenicity, and evading ubiquitination to ultimately improve transduction efficiency of retinal pericytes. TRANSLATIONAL RELEVANCE: Identification of novel retinal pericyte targeting rAAV vectors enables the development of new, long-lasting gene therapies for retinal diseases such as diabetic retinopathy and wet age-related macular degeneration.

Animals↗
PubMed1991

Directionality of evolution at molecular and organismic levels.

The molecular evolution theories of Eigen and Kimura are compared and their difference is explained. In terms of Eigen's theory for the evolution of macromolecules, the selection of genotypes occurs directly. The physical meaning of the neutral theory is the degeneracy of the correlation between a phenotype and a genotype at the molecular level. A model theory of evolution on a fitness landscape is proposed. The theory shows that the constraints of selection determined by the structure and dynamics of previous evolution stages increases its rate strongly.

Biological Evolution↗
PubMed1987

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins.

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.

Animals↗