Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA.
Figure 1: Genome editing approaches for altering the genetic code on a genome-wide scale in E. Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, H-6726, Hungary.
Pleiotropy occurs when a single gene influences multiple phenotypic traits that are seemingly unrelated. An extreme form of mistranslation in which a codon can be translated as two different amino acids.
Evolution of similar phenotypes in different populations or species as a result of adaptation to similar environments or ecological niches. Interactions between two mutations whereby the phenotypic effect of one mutation depends on the presence of another mutation. Modification of the genetic information encoded by the genome using in vivo, directed modification (such as replacement, removal or insertion of DNA bases) of a single locus or multiple loci. Genomes that carry only the minimal genetic information necessary for life in a given environmental condition. A recombination engineering system that allows efficient manipulation of genomic DNA at predetermined locations.
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA. Supplementary Figure 2: Design of PCR primers for genotyping of knock-in mutations in rat genome. This video demonstrates the microinjection of the CRISPR-Cas system into the pronucleus of a rat zygote fixed with a holding pipette on the left. In the error-prone NHEJ pathway, the ends of a DSB are processed by endogenous DNA repair machinery and rejoined, which can result in random indel mutations at the site of junction.

His group focuses on evolutionary systems biology, using microbial selection experiments, genome engineering and theoretical modelling.
In the case of antagonistic pleiotropy, expression of the pleiotropic gene has mixed, competing effects; some of these are beneficial but others are detrimental to the organism. As the size of the investigated system and the number of corresponding parameters increase, the number of combinations that one has to examine grows exponentially, which requires an intolerable amount of time to examine them. It uses synthetic oligonucleotides and a range of accessory tools, including engineered nucleases, and DNA repair and recombination enzymes.
Reduction towards a minimal essential gene set can occur either naturally (for example, in symbionts) or by genetic engineering.
A highly efficient genome editing method that can generate a large and heterogeneous population of mutant bacterial genomes within days. It does not require extensive sequence similarity and relies on site-specific recombinases that catalyse reciprocal recombination of DNA at short sequences. Owing to limitations in the length of DNA that is amenable to direct chemical synthesis, construction of synthetic chromosomes is a hierarchical process, in which synthetic oligonucleotides are assembled into larger DNA segments in a step-wise manner using in vitro and in vivo assembly methods.
Gersbach is in the Department of Biomedical Engineering and at the Center for Genomic and Computational Biology, Duke University, and in the Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.
NHEJ promotes direct ligation of the DSB ends in an error-prone manner, usually leading to indels (small insertions and deletions).
After the incubation, the protein was discarded and the DNA was analyzed by agarose gel electrophoresis.
An obvious pronuclear swelling is shown to demonstrate the successful pronuclear injection. Indel mutations occurring within the coding region of a gene can result in frameshifts and the creation of a premature stop codon, resulting in gene knockout.
Conjugative assembly genome engineering (CAGE) allows step-wise transfer of individually engineered, marked genomic modules into a single genome. Using oligonucleotide-mediated allelic replacement technology in a cyclic and automated manner, MAGE can simultaneously target and modify multiple genomic locations across a large population of cells. HR requires a homologous sequence, either from a plasmid or from single-stranded oligonucleotides (ssODN), to serve as a donor template for repair of both broken strands in a high-fidelity manner.
Alternatively, a repair template in the form of a plasmid or ssODN can be supplied to leverage the HDR pathway, which allows high fidelity and precise editing.

Full transfer of a genomic segment is controlled by inserting oriT conjugational start sites and selection markers at appropriate positions (not shown). As a postdoctoral researcher, he studied protein evolution, evolution of metabolic and genetic interaction networks, and co-evolution of bacteria with phages. The other primer is located in rat genome outside the homology arms (left arm or right arm).
The PCR products were amplified with the primers outside of the homology region indicated in (a). He has worked at the University of Bath, UK, at the European Molecular Biology Laboratory, Heidelberg, Germany, and at the University of Oxford, UK. About 400 bp extension of both left and right arm of the donor plasmid can be used as the positive control for testing the primers (optional). All occurrences of the UAG codon have been removed, and translation termination at UAG has been eliminated by deleting prfA, which is the open reading frame (ORF) encoding release factor 1 (RF1). The now blank UAG codon has been reintroduced, along with an orthogonal set of aminoacyl-tRNA synthase and tRNA, to encode a non-standard amino acid. Before establishing a research group in Szeged, he worked at the University of Bath, the University of Manchester and the University of Cambridge, all of which are in the UK. A genetic correction of disease-causing mutation associated with ?-1 antitrypsin deficiency is shown here. His main interest lies in using computational systems biology models to address evolutionary questions and to understand the biochemical architecture of the genotype–phenotype map. He worked as a postdoctoral fellow at the McArdle Laboratory for Cancer Research at the University of Wisconsin-Madison, USA. Since 1996, his laboratory at the Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary, focuses on microbial genomics and has a special interest in engineering streamlined bacterial genomes.

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