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This volume details the fundamentals of the CRISPR-Cas system, and its protocols illustrate advances in CRISPR-Cas techniques for efficient genome editing. Introductory chapters provide a wide horizon of CRISPR/Cas-based methods and applications. Additional chapters guide readers through HDR-mediated editing, sgRNA design, the step-by-step procedure of multiplex adenine base editing experiments in rice, generating mutants for rice, wheat, Brachypodium, Barley, Flax, and Phytophthora, visual screening of mutants, gene deletion (knock-out), tagging (knock-in) in mammalian cells, the cloning-free (DNA-free) technique, cell-penetrating peptides, generating a genome-edited banana, and nuclear genome editing of Chlamydomonas employing CRISPR-Cpf1 combined with a single-stranded DNA (ssODN) repair template.
Authoritative and cutting-edge, CRISPR-Cas Methods aims to assist researchers who are new to the field and are aiming to learn how best to adopt this technology for a particular organism.
List of contents
1. Efficient Homologous Recombination Mediated in Planta Gene Targeting by Egg-cell Specific Expression of Staphylococcus aureus Cas9 from Arabidopsis.- 2. Rice gene knockout or down regulation through CRISPR-Cas9.- 3. Simultaneous CRISPR/Cas-mediated Single Base Editing in Rice Plant at More than One Locus .- 4. CRISPR-Cas9 Mediated Genome Editing of the Model Grass Species Brachypodium distachyon.- 5. CRISPR/Cas9-mediated Gene Editing of the Plant Pathogenic Oomycete Phytophthora palmivora.- 6. An effective CRISPR/Cas9 technology for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species.- 7. An optimized RNA-guided Cas9 system for efficient simplex and multiplex genome editing in barley.- 8. Genome editing of mammalian cells using CRISPR/Cas: From in silico designing to in-culture validation.- 9. Cloning free (DNA-free) CRISPR/Cas9-mediated gene knockout in human liver cell line and its detection.- 10. A procedure to design guideRNA, assembly fragments, and detect mutation for genome editing in flax.- 11. Delivery of Cas9/sgRNA RNP into wheat microspores using synthetic CPP for genome editing and gene expression modulation.- 12. CRISPR- Cas9 mediated gene editing in wheat: a step by step protocol.- 13. CRISPR-Cas9 based genome editing of banana.- 14. Homology-directed transgene-free gene editing in Chlamydomonas reinhardtii.
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