Crop Genome Editing Using CRISPR/Cas9

1. Introduction to genome editing• ZFN• TALENs• CRISPR/Cas92. Crop improvement using CRISPR/Cas93. 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 transcription5. 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 colonies6. Protoplast Isolation from plants• Protoplast isolation by enzymatic digestion using cellulases, hemicellulases and pectinases• Protoplast cell count by haemocytometer• Protoplast viability test by fluorescent staining7. 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 events8. Agrobacterium mediated transformation of CRISPR constructs for the generation of stable mutant plants9. Recent Advancements in CRISPR/Cas9 genome editing in plants