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Elsevier

Crop Genome Editing Using CRISPR/Cas9

Theory and Practice

  • 1st Edition - December 1, 2019
  • Latest edition
  • Authors: Ramalingam Ravindhran, Govindan Ganesan, A. Vinoth
  • Language: English

Crop Genome Editing Using CRISPR/Cas9: Theory and Practice is a highly useful reference for implementing genome engineering technologies, particularly CRISPR related projects… Read more

Description

Crop Genome Editing Using CRISPR/Cas9: Theory and Practice is a highly useful reference for implementing genome engineering technologies, particularly CRISPR related projects in agricultural crops and other plants. This book provides an introduction to CRISPR’s basic science and applied aspects, along with detailed protocols. It presents a detailed workflow, beginning with genome sequence retrieval and then mutation analysis in genome edited events using sequencing tools. The book helps those in the field methodically plan, design and conduct experiments. This practical guide will dramatically help researchers in accelerating conventional plant breeding programs.

Key features

  • Offers a detailed review of literature on genome editing tools, with special emphasis paid to CRISPR/Cas9 and its advancements
  • Contains step-by-step guidelines for single guide RNA design, CRISPR vector construction, protoplast transformation, mutation analysis and Agrobacterium-based regeneration of mutant plants
  • Includes detailed troubleshooting tips during various steps

Readership

Graduate students, Postdoctoral fellows, scientists, professors in plant and crop science.

Table of contents

1. Introduction to genome editing
ZFN
TALENs
CRISPR/Cas9


2. Crop improvement using CRISPR/Cas9


3. Designing of sgRNA
Sequence retrieval from NCBI
Exon-Intron prediction in plant genes using FGenesh software
Single guide RNA (sgRNA) design using CRISPR-P software


4. Tools and Resources for the construction of CRISPR vectors
CRISPR plasmids with Cas9 variants
Promoters for sgRNA transcription
In vitro sgRNA transcription


5. Clonin g of CRISPR constructs
Cloning of sgRNAs into CRISPR vectors by restriction digestion and ligation
Construction of CRISPR vectors with multiple sgRNAs by restriction digestion and ligation
E. coli transformation and Colony PCR of transformants for identification of sgRNA clones
Plasmid isolation from transformed colonies


6. Protoplast Isolation from plants
Protoplast isolation by enzymatic digestion using cellulases, hemicellulases and pectinases
Protoplast cell count by haemocytometer
Protoplast viability test by fluorescent staining


7. Delivery of CRISPR constructs into protoplasts and mutation analysis
PEG-mediated transformation in protoplasts
Protoplast DNA isolation
Mutation analysis by PCR and T7 endonuclease assay
DNA Sequence analysis of Genome Edited events


8. Agrobacterium mediated transformation of CRISPR constructs for the generation of stable mutant plants


9. Recent Advancements in CRISPR/Cas9 genome editing in plants

Product details

  • Edition: 1
  • Latest edition
  • Published: December 1, 2029
  • Language: English

About the authors

RR

Ramalingam Ravindhran

Professor and Head, Department of Plant Biology and Biotechnology, Loyola College, Chennai, India
Affiliations and expertise
Professor and Head, Department of Plant Biology and Biotechnology, Loyola College, Chennai, India

GG

Govindan Ganesan

Research Assistant Professor, Department of Genetic Engineering, SRM University, Kattankulathur, India
Affiliations and expertise
Research Assistant Professor, Department of Genetic Engineering, SRM University, Kattankulathur, India

AV

A. Vinoth

Assistant Professor, Department of Botany, St. Xavier’s College, Tamil Nadu, India
Affiliations and expertise
Assistant Professor, Department of Botany, St. Xavier’s College, Tamil Nadu, India