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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.