This second volume provides new and updated methods detailing advancements in CRISPR-Cas technical protocols. Chapters guide readers through protocols on prime editing, base editing, multiplex editing, editing in cell-free extract, in silico analysis of gRNA secondary structure and CRISPR-diagnosis.

Authoritative and cutting-edge, CRISPR-Cas Methods, Volume 2 aims to serves as a laboratory manual providing scientists with a holistic view of CRISPR-Cas methodologies and its practical application for the editing of crop plants, cell lines, nematode and microorganism.

The chapter CRISPR/Cas9-mediated gene editing in human induced pluripotent stem cells is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
A Complete Methodology for the Instruction of CRISPR-based Gene Editing Using a Simplified Cell-free Extract System with Genetic Readout in Bacteria.- In silico Analysis of gRNA Secondary Structure to Predict Its Efficacy for Plant Genome Editing.- Efficient Multiplexed CRISPR-Cas12a Genome Editing in Plants.- CRISPR-dCas9 Based Targeted Manipulation of DNA Methylation in Plants.- Rapid Assembly of Multiplex Natural CRISPR Arrays.- Assembly and Assessment of Prime Editing Systems for Precise Genome Editing in Plants.- Designing, Performing and Analysing CRISPR-Cas9-mediated Genome Editing Experiments in Leguminous Plant.- Generation of Knockout and Fragment Deletion Mutants in Soybean by CRISPR-Cas9.- Targeted Base Editing in Soybean using a CRISPR-Cas9 Cytidine Deaminase Fusion.- Genome Editing in Potato Using a Hairy Root Transformation System.- Efficient CRISPR-Cas9-Mediated Genome Editing in Tomato.- CRISPR-Cas9-Mediated Genome Editing in Rice: A Systematic Protocol for Single and Multi-Target Vector Construction.- Generating Clonal Seeds from Hybrid Rice with CRISPR-Cas9.- CRISPR-Cas12a-based DNA Detection for Fast Pathogen Diagnosis and GMO Test in Plants.- CRISPR-Cas9-mediated Gene Editing in Human Induced Pluripotent Stem Cells.- Cell-Type-Specific CRISPR-Cas9 System with miRNAs.- CRISPR-Cas9 gene Editing in Mammalian Cells Using LentiCRISPRv2/LentiGuide-puro Vectors.- An Approach to Proximity Ligation by T4 RNA Ligase to Screen sRNA that Regulate CRISPR-Cas systems.- Genetic Engineering of a Phage-based Delivery System for Endogenous III-A CRISPR-Cas System against Mycobacterium tuberculosis.- CRISPR-Cas9-Mediated Genome Editing in Escherichia coli Bacteriophages.- CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum.- Gene Targeting in Caenorhabditis elegans Using a Combination of Multiple sgRNAs and a Homologous Recombination-mediated Repair.- Design of Repair Templates for CRISPR-Cas9-triggered Homologous Recombination in Caenorhabditis elegans.