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.

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.".