Format:
Online-Ressource (xx, 486 S.)
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graph. Darst., Ill.
ISBN:
9780128014158
Series Statement:
Methods in enzymology 547
Content:
This new volume of Methods in Enzymologycontinues the legacy of this premier serial with quality chapters authored by leaders in the field.
Content:
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. Methods to assess mitochondrial function is of great interest to neuroscientists studying chronic forms of neurodegeneration, including Parkinson's, Alzheimer's, ALS, Huntington's and other triplet repeat diseases, but also to those working on acute conditions such as stroke and traumatic brain injury. This volume covers research methods on how to assess the life cycle of mitochondria including trafficking, fusion, fission, and degradation. Multiple persp
Note:
Description based upon print version of record
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Front Cover; The Use of CRISPR/Cas9, ZFNs, and TALENs in Generating Site-Specific Genome Alterations; Copyright; Contents; Contributors; Preface; Chapter One: In Vitro Enzymology of Cas9; 1. Introduction; 2. Expression and Purification of Cas9; Day 1: Cell transformation; Day 2: Culture growth and induction; Day 3: Cas9 purification by IMAC; Day 4: IEX and SEC chromatographic steps; Day 5: Concentration and storage; 3. Preparation of Guide RNAs; Day 1: Preparation of transcription template; Day 2: In vitro transcription and gel purification; Day 3: Gel purification-continued
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4. Endonuclease Cleavage AssaysSubstrate preparation; Cleavage assay; Interpretation of cleavage assays; 5. Concluding Remarks; Acknowledgments; References; Chapter Two: Targeted Genome Editing in Human Cells Using CRISPR/Cas Nucleases and Truncated Guide RNAs; 1. Introduction; 2. Methods; 2.1. Identification of target sites using ZiFiT; Required materials; Ensure query sequence is valid; Design target sites; 2.2. Construction of tru-gRNA expression plasmids; 2.2.1. Reagents; 2.2.2. Protocol; 2.3. Transfection of sgRNA and Cas9 expression plasmids into human cells; 2.3.1. Reagents
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2.3.2. Protocol2.3.2.1. Prior to Day 1; 2.4. Quantitative T7EI assays to assess frequencies of targeted genome editing; 2.4.1. Reagents; 2.4.2. Protocol; Conflict of Interest; References; Chapter Three: Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes; 1. Introduction; 1.1. Introduction to programmable nucleases for genome editing; 1.2. Overview of methods to study specificity of genome-editing agents; 1.2.1. Discrete off-target site testing; 1.2.2. Genome-wide selections; 1.2.3. Minimally biased selections in vitro and in cells
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1.3. Insights and improvements from ZFN specificity studies1.4. Insights and improvements from TALEN specificity studies; 1.5. Insights and improvements from Cas9 specificity studies; 2. Methods; 2.1. Overview of in vitro selection-based nuclease specificity profiling; 2.2. Pre-selection library design; 2.3. In vitro selection protocol; 2.3.1. Before Day 1: Design and synthesize pre-selection library oligonucleotides; 2.3.2. Day 1: Circularize library oligonucleotides; 2.3.3. Day 2: Confirm circularization of library oligonucleotides and perform rolling-circle amplification
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2.3.4. Day 3: Quantify and digest pre-selection library2.3.5. Day 4: PCR of pre- and post-selection libraries; 2.3.6. Day 5: High-throughput sequencing and analysis; 2.4. Confirmation of in vitro-identified genomic off-target sites; 3. Conclusion; Acknowledgments; References; Chapter Four: Genome Engineering with Custom Recombinases; 1. Introduction; 2. Target Identification; 3. Recombinase Construction; 4. Measurements of Recombinase Activity; 4.1. Reporter plasmid construction; 4.2. Luciferase assay; 5. Site-Specific Integration; 5.1. Donor plasmid construction; 5.2. Cell culture methods
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5.2.1. PCR confirmation of integration
Additional Edition:
ISBN 9780128016152
Additional Edition:
Print version Mitochondrial Function
Language:
English
Subjects:
Biology
Keywords:
Mitochondrium
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