Main description:

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.

Contents:

1. Homing Endonucleases: From Genetic Anomalies to Programmable Genomic Clippers

Marlene Belfort and Richard P. Bonocora

2. Bioinformatic Identification of Homing Endonucleases and Their Target Sites

Eyal Privman

3. PCR-Based Bioprospecting for Homing Endonucleases in Fungal Mitochondrial rRNA Genes

Mohamed Hafez, Tuhin Kumar Guha, Chen Shen, Jyothi Sethuraman, and Georg Hausner

4. Mapping Homing Endonuclease Cleavage Sites Using In Vitro Generated Protein

Richard P. Bonocora and Marlene Belfort

5. Mapping Free-Standing Homing Endonuclease Promoters Using 5'RLM-RACE

Ewan A. Gibb

6. PCR Analysis of Chloroplast Double-Strand Break (DSB) Repair Products Induced by I-CreII in Chlamydomonas and Arabidopsis

Taegun Kwon, Obed W. Odom, Weihua Qiu, and David L. Herrin

7. A Two-Plasmid Bacterial Selection System for Characterization and Engineering of Homing Endonucleases

Ning Sun and Huimin Zhao

8. Rapid Screening of Endonuclease Target Site Preference Using a Modified Bacterial Two-Plasmid Selection

Jason M. Wolfs, Benjamin P. Kleinstiver, and David R. Edgell

9. A Yeast-Based Recombination Assay for Homing Endonuclease Activity

Jean-Charles Epinat

10. Rapid Determination of Homing Endonuclease DNA Binding Specificity Profile

Lei Zhao and Barry L. Stoddard

11. Quantifying the Information Content of Homing Endonuclease Target Sites by Single Base Pair Profiling

Joshua I. Friedman, Hui Li, and Raymond J. Monnat, Jr.

12. Homing Endonuclease Target Site Specificity Defined by Sequential Enrichment and Next-Generation Sequencing of Highly Complex Target Site Libraries

Hui Li and Raymond J. Monnat, Jr.

13. Homing Endonuclease Target Determination Using SELEX Adapted for Yeast Surface Display

Kyle Jacoby and Andrew M. Scharenberg

14. Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

Sarah K. Baxter, Andrew M. Scharenberg, and Abigail R. Lambert

15. Bioinformatics Identification of Coevolving Residues

Russell J. Dickson and Gregory B. Gloor

16. Identification and Analysis of Genomic Homing Endonuclease Target Sites

Stefan Pellenz and Raymond J. Monnat, Jr.

17. Redesigning the Specificity of Protein-DNA Interactions with Rosetta

Summer Thyme and David Baker