In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.

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The green fluorescent protein

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

/pdf/programmable-editing-of-a-target-base-in-genomic-dna-without-53ykhw8ya8.pdf

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2) However, slow maturation is still a big obstacle to the use of GFP variants for visualization These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-) For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired Here we describe the development of an improved version of YFP named "Venus" Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl- We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells Its secretion was readily monitored by measuring release of fluorescence into the medium The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications

Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

Progress and problems with the use of viral vectors for gene therapy

The use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Biocatalytic processes can now be carried out in organic solvents as well as aqueous environments, so that apolar organic compounds as well as water-soluble compounds can be modified selectively and efficiently with enzymes and biocatalytically active cells. As the use of biocatalysis for industrial chemical synthesis becomes easier, several chemical companies have begun to increase significantly the number and sophistication of the biocatalytic processes used in their synthesis operations.

Industrial biocatalysis today and tomorrow

DNA SHUFFLING is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly1. Computer simulations called genetic algorithms2–4have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis5–11 and error-prone PCR12,13 are not combinatorial and thus are limited in searching sequence space1,14. We have tested mutagenic DNA shuffling for molecular evolution14–18 in a p-lactamase model system9,19. Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 μg ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase9.

Rapid evolution of a protein in vitro by DNA shuffling.

Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.

https://www.pnas.org/content/pnas/91/22/10747.full.pdf

DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Improved green fluorescent protein by molecular evolution using DNA shuffling.

DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination, combining useful mutations from individual genes Libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction Template switching causes crossovers in areas of sequence homology Our previous studies used single genes and random point mutations as the source of diversity An alternative source of diversity is naturally occurring homologous genes, which provide 'functional diversity' To evaluate whether natural diversity could accelerate the evolution process, we compared the efficiency of obtaining moxalactamase activity from four cephalosporinase genes evolved separately with that from a mixed pool of the four genes A single cycle of shuffling yielded eightfold improvements from the four separately evolved genes, versus a 270- to 540-fold improvement from the four genes shuffled together, a 50-fold increase per cycle of shuffling The best clone contained eight segments from three of the four genes as well as 33 amino-acid point mutations Molecular breeding by shuffling can efficiently mix sequences from different species, unlike traditional breeding techniques The power of family shuffling may arise from sparse sampling of a larger portion of sequence space

DNA shuffling of a family of genes from diverse species accelerates directed evolution

http://mcb.berkeley.edu/labs/krantz/pdf/gene_synthesis/stemmer-gene-1995.pdf

Willem P. C. Stemmer

Papers

Rapid evolution of a protein in vitro by DNA shuffling.

DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.

Improved green fluorescent protein by molecular evolution using DNA shuffling.

DNA shuffling of a family of genes from diverse species accelerates directed evolution

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.