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Homing Endonucleases: Methods and Protocols aims at providing molecular biologists with a comprehensive resource to identify and characterize homing endonucleases from genomic sequence, to deduce the biological basis of binding and cleavage specificity, as well as to provide protocols to redesign endonuclease target specificity for genome-editing applications. Engineering of designer homing endonucleases has set the stage for genome editing of complex eukaryotic genomes with a broad range of potential applications including targeted gene knockouts in model organisms and gene therapy in humans, making this book a valuable resource for future research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Homing Endonucleases: Methods and Protocols serves as a key reference for all labs studying site-specific DNA endonucleases.
List of contents
Homing Endonucleases: From Genetic Anomalies to Programmable Genomic Clippers.- Bioinformatic Identification of Homing Endonucleases and Their Target Sites.- PCR-Based Bioprospecting for Homing Endonucleases in Fungal Mitochondrial rRNA Genes.- Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein.- Mapping Free-Standing Homing Endonuclease Promoters Using 5'RLM-RACE.- PCR Analysis of Chloroplast Double-Strand Break (DSB) Repair Products Induced by I-CreII in Chlamydomonas and Arabidopsis.- A Two-Plasmid Bacterial Selection System for Characterization and Engineering of Homing Endonucleases.- Rapid Screening of Endonuclease Target Site Preference Using a Modified Bacterial Two-Plasmid Selection.- A Yeast-Based Recombination Assay for Homing Endonuclease Activity.- Rapid Determination of Homing Endonuclease DNA Binding Specificity Profile.- Quantifying the Information Content of Homing Endonuclease Target Sites by Single Base Pair Profiling.- Homing Endonuclease Target Site Specificity Defined by Sequential Enrichment and Next-Generation Sequencing of Highly Complex Target Site Libraries.- Homing Endonuclease Target Determination Using SELEX Adapted for Yeast Surface Display.- Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes.- Bioinformatics Identification of Coevolving Residues.- Identification and Analysis of Genomic Homing Endonuclease Target Sites.- Redesigning the Specificity of Protein-DNA Interactions with Rosetta.
Summary
Homing Endonucleases: Methods and Protocols aims at providing molecular biologists with a comprehensive resource to identify and characterize homing endonucleases from genomic sequence, to deduce the biological basis of binding and cleavage specificity, as well as to provide protocols to redesign endonuclease target specificity for genome-editing applications. Engineering of designer homing endonucleases has set the stage for genome editing of complex eukaryotic genomes with a broad range of potential applications including targeted gene knockouts in model organisms and gene therapy in humans, making this book a valuable resource for future research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Homing Endonucleases: Methods and Protocols serves as a key reference for all labs studying site-specific DNA endonucleases.
